Synthetic heat-stable enterotoxin polypeptide of Escherichia coli and multimers thereof

ABSTRACT

A synthetic polypeptide having at least about 10% of the immunological activity of biologic heat-stable enterotoxin of E. coli. The synthetic polypeptide includes at least 14 amino acids in the sequence, from amino-terminus to carboxy-terminus, represented by the formula: CysCys-GluLeuCysCysTyr(Asn)ProAlaCysAla(Thr)GlyCysAsn(Tyr) wherein the amino acid in parentheses may replace the immediately preceding amino acid residue, and at least one intramolecular disulfide bond formed between the Cys residues. The Cys residues that are not part of the intramolecular disulfide bond can be replaced by other amino acid residues or be bonded to substituent moieties. The polypeptides can be a monomeric or multimeric material containing an intramolecular, intrapolypeptide and/or an intramolecular, interpolypeptide cystine disulfide bond.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of copending application Ser. No.455,265, filed Jan. 3, 1983, now U.S. Pat. No. 4,545,931.

TECHNICAL FIELD

The present invention relates to a synthetic polypeptide correspondingto heat-stable enterotoxin of Escherichia coli, and more particularly tosynthetic polypeptides and multimers therof that comprise principaldeterminant domains responsible for the antigenicity of the E. coliheat-stable enterotoxin.

BACKGROUND ART

Acute diarrheal disease due to transient colonization of the small bowelby enterotoxigenic strains of Escherichia coli (E. coli or ETEC) is amajor health problem of global scope for both humans and for animalhusbandry. These organisms, together with rotavirus, are the principalcause of the often fatal acute diarrhea that is common among infantsliving in underdeveloped countries and among neonatal animals,particularly lambs and piglets. ETEC strains are also the usual cause ofacute diarrhea among persons from temperate zones who travel to thetropics, and may be responsible for sporadic or epidemic episodes ofdiarrhea among children and adults living in either temperate ortropical areas.

The disease caused by ETEC is mediated by the release of twoenterotoxins, either singly or together. The large molecular weight,antigenic heat-labile toxin (LT) has been purified to homogeneity andits subunit structure characterized as five B subunits which attach theholotoxin to the specific GM₁ ganglioside receptors on the mucosalsurface, and a single A subunit which stimulates intracellular adenylatecyclase activity, thus evoking fluid and electrolyte secretion.

The low molecular weight, heat-stable toxin (ST) produced by ETECstrains of human or porcine origin has also recently been purified.Preparations of ST have a relatively high content of half-cystine, causesecretion by stimulating guanylate cyclase and are haptenic as evidencedby their capacity to raise an antitoxin response in animals immunizedwith the toxin coupled to a large molecular weight carrier.

The most practical approach for the prevention of ETEC-induced diarrheawould be an immunization program that provides protection againstheterlogous ETEC serotypes that produce either or both of the LT or STenterotoxins. Immunization with either the biologic LT or the biologicST toxin evokes an antitoxin response in experimental animals thatprotects against homologous and heterlogous serotypes of strains thatproduce the specific toxin used for immunization. Immunization with theLT whole toxin or its B subunit yields protection against viableheterlogous strains that produce this toxin alone (LT⁺ /LT⁻) or togetherwith ST (LT⁺ /ST⁺), but not against those which make just ST (LT⁻ /ST⁺).

Immunization with biologic ST coupled to a large molecular weightcarrier arouses serum antibodies that passively neutralize the secretoryeffect in the suckling mouse model of ST produced by heterlogousstrains. Immunization also provides protection against direct challengewith viable heterlogous LT⁻ /ST⁺, but not LT-producing strains. Neitherof these toxins is suitable for immunization when given along, however,in view of their toxicity, their failure to provide protection againststrains which produce the other toxin form, and the fact that the largemolecular weight carriers that have been used to render the haptenicbiologic ST molecule antigenic are unsuitable for human use.

Klipstein et al., Infect. Immun., 37: 550-557 (1982) have reported thedevelopment of a vaccine made by conjugating the biologic ST toxin tothe LT toxin by means of the carbodiimide reaction. As a result of thatreaction, biologic ST acquires antigenicity when coupled to the largemolecular weight LT carrier, while both cross-linked toxins retain mostof ther antigenicity but loose most of their toxix properties. Ratsimmunized with the vaccine so produced were strongly protected againstchallenge with either LT or biologic ST and with viable ETEC strainswhich produce those toxins.

A semi-pure preparation of biologic ST was used for that vaccine becauseof the relatively low yield of pure biologic ST obtained by theavailable purification techniques which involve multiple chromatographicseparation steps. The inclusion of the heterogeneous material in thevaccine may preclude its use for human immunization, however.

The present invention, relating to the synthetically produced ST, hasovercome the problem of using ST derived from natural sources in thatsynthetic ST can be made in large quantities and in purified form, andhas properties similar to those described for pure ST obtained bybacterial growth of a human ETEC strain. [Staples et al., J. Biol.Chem., 255: 4716-4721 (1980); and Chan et al., J. Biol. Chem., 256:7744-7746 (1981)].

At least two types of ST have been identified by their physicalproperties. The first type known as ST I (also referred to as STa) issoluble in methanol and is active in the suckling mouse model. Thesecond type, ST II (also referred to as STb) is methanol insoluble andnot active in the suckling mouse model, but is active in ligated pigileal loops.

Among the ST I polypeptides, at least three similar polypeptides, ordeterminant domains of those polypeptides, have been identified, andtheir amino acid sequences determined. These three types of ST I arereferred herein as (i) ST Ia which was initially found in a bovine E.Coli strain and a portion of which is also encoded in porcine strains,(ii) that designated ST Ib from a human isolate of E. coli and (iii) STIc also isolated from human-infecting E. coli.

The nucleotide sequence coding for the ST Ia polypeptide has beendetermined. Translation of the nucleotide sequence into a polypeptideamino acid sequence leads to a polypeptide that contains 72 amino acidscapped at the carboxy-terminus with a tyrosine group [So et al., Proc.Natl. Acad. Sci. USA, 77: 4011-4015 (1980)]. The ST Ic polypeptide isthough to also contain 72 amino acids as well as several homologousdomains with the ST Ia polypeptide. The ST Ib polypeptide is reported tocontain only 18 amino acids ([Chan et al., J. Biol. Chem., 256:7744-7746 (1981)].

The 18 amino acids of the ST Ib polypeptide (18-mer) show great homologyto amino acids 55 through 72 for the polypeptide of ST Ia. Thehomologous, almost identical, region is illustrated hereinbelow,beginning at amino acid number 55, from left to right and in thedirection of amino-terminus to carboxy-terminus, of the ST Iapolypeptide:

ST Ia: AsnThrPheTyrCysCysGluLeuCysCys

ST Ib: AsnThrPheTyrCysCysGluLeuCysCys

AsnProAlaCysAlaGlyCysTyr

TyrProAlaCysAlaGlyCysAsn

More recent reports by Takeda et al., Abstracts, 19th Joint ConferenceUS-Japan Cooperative Medical Science Program, Cholera Panel, 87-88(1983) and Ikemura et al., Chem. Letters, (Chem. Soc. Japan), 101-104(1983) have indicated the presence of further polypeptide sequences forthis 18-mer polypeptide. Those workers referred to the ST moleculeobtained from human and porcine strains of ETEC as ST_(h) and ST_(p),respectively. The amino acid residue sequences reported by thoseworkers, from left to right and in the direction from amino-terminus tocarboxy-terminus, are:

ST_(h) AsnSerSerAsnTyrCysCysGluLeuCysCys

ST_(p) - - - AsnThrPheTyrCysCysGluLeuCysCys

AsnProAlaCysThrGlyCysTyr

AsnProAlaCysAlaGlyCysTyr

As can be seen from a comparison of both of the above sets of sequences;i.e., ST Ia, ST Ib, ST_(h) and ST_(p), a great deal of homology isshared among the carboxy-terminal fourteen residues of each of the foursequences shown.

Those workers also reported a synthesis of ST_(h). Solution methods ofsynthesis were used to prepare the blocked polypeptide. Blocking groupswere removed with hydrogen fluoride, and the Cys mercapto groups(thiols) were air oxidized. Air oxidation was carried out at apolypeptide concentration of 10⁻⁵ molar in distilled water adjusted to apH value of 8.0 with aqueous ammonia. Oxidation was continued until freethiol groups disappeared.

Biologic activity of the synthetic ST_(h) in a suckling mouse assay wasreported to be the same as that for native toxin. Toxicity of thesynthetic material was reported to be neutralized by antisera againstthe native toxin.

Examination of the above four 18-amino acid polypeptide sequences alsoreveals that six half-cystine (Cys) residues that are present. Oxidationof those half-cystine residues to cystine residues containingintramolecular disulfide bonds in the naturally occuring enterotoxin isthought to lend the observed heat stability to that material.

It is further noted, however, that while cystine disulfide bonds areknown to be present in biologic ST, it is not known which pairs ofhalf-cystine residues combine to form the three disulfide bonds that arepresent in the native ST molecule. Those three sulfide bonds cantheoretically be formed from fifteen different combinations of the sixCys residues present.

Staples et al., supra, have shown that the disulfide linkages ofbiologic ST are required for biological activity of the toxin. Thus,chemical reduction to form half-cystines or performic acid oxidation tocysteic acid was shown to destroy the biological activity of the toxin.In addition, Chan et al., supra, have reported that the first fourresidues from the amio-terminus of the homologous 18-amino acids of theabove sequence of St Ib are not required for biological activity. Thus,biological activity was obtained from the amino acid-containingpolypeptide comprising the above carboxy-terminal 14 amino acids andtheir disulfide bonds.

Aimoto et al, Biochem. Biophys. Res. Chem., 112: 320-326 (Apr. 15, 1983)have reported on the synthesis of the carboxy-terminal fourteen aminoacid residues of the beforedescribed ST_(h). That synthetic molecule wasreported to have biologic activity 2-5 times that of the native ST_(h)on a molar basis, using a suckling mouse assay.

In an oral presentation on Aug. 29, 1982 by Duflot et al., ProceedingsEuropean Peptide Symposium: 683-686, published in Berlin in June of1983, those workers reported the synthesis of a porcine and human ST18-mer polypeptides having their Cys mercapto groups blocked (S-blocked)with acetamidomethyl groups. Those amino acid residue sequences werepurportedly identical to the sequences reported by So et al, supra, forST Ia and by Chan et al., supra, for ST Ib. However, the seventh aminoacid residue from the amino-terminus of the sequences reported by Duflotet al. was a glycine residue (Gly), while that residue in thebeforedescribed sequences is a glutamic acid residue (Glu).

Duflot et al. reported that immunization of micr or rabbits with theirS-blocked porcine ST toxin coupled to tetanus toxoid or ovalbuminproduced antibodies that recognized the natural or the synthetic toxinsequally. Substantially no biologic activity in the suckling mouse assaywas reported for the S-blocked, porcine, synthetic polypeptide toxin.Those authors reported the lack of biologic activity to be due to theabsence of intramolecular disulfide bonds in the S-blocked molecule,which is in keeping with the prior report of Staples et al., supra.

SUMMARY OF THE INVENTION

The present invention contemplates a synthetic polypeptide having anantigenicity, as a free monomer or as a multimer, that is at least about10 percent of that of biologic E. coli heat-stable enterotoxin (ST). Thesynthetic polypeptide includes the amino acid residue sequence, takenleft to right and in the direction from amino-terminus tocarboxy-terminus, represented by the formula: ##STR1##

wherein the three specific amino acid residues in parentheses are eachan alternative to the immediately preceding amino acid residue in thesequence;

a-f and g-l are integers each having a value of zero or one, with theproviso that if the value of any of a-f or g-l is zero, thecorresponding R_(a-f) ¹⁻⁶ - or R_(g-l) ⁷⁻¹² -group is absent, and whenan R_(a-f) ¹⁻⁶ -group is absent the sulfur atom of the Cys residuehaving an absent R_(a-f) ¹⁻⁶ -group forms a cystine disulfide bond,while if the value of the a-f or g-l is one, the corresponding R_(a-f)¹⁻⁶ - or R_(g-l) ⁷⁻¹² -group is present;

the R_(a-f) ¹⁻⁶ -groups, when taken individually, are the same ordifferent moieties bonded to the sulfur atom of the Cys residues and areselected from the group consisting of hydrogen, an alkyl groupcontaining 1 to about 4 carbon atoms, a substituted alkyl groupcontaining 2 to about 20 carbon atoms, an acyl group containing 1 toabout 8 carbon atoms, and a substituted acyl group containing 2 to about10 carbon atoms;

the R_(g-l) ⁷⁻¹² -groups are the same or different alternative aminoacid residues to each immediately preceding Cys residue;

at least two of a-f and two of g-l are zero and two Cys residues arepresent with the proviso that the synthetic polypeptide contains atleast one intramolecular cystine disulfide bond formed from the at leasttwo Cys residues present; and

"m" is an integer having the value of zero or one with the proviso thatif "m" is zero R_(m) ¹³ is absent, and if "m" is one R_(m) ¹³ isselected from the group consisting of a chain containing 1 to about 54amino acid residues, a linking group, and the acyl portion of acarboxylic acid containing 1 to about 20 carbon atoms forming an amidebond with the amine of the amino-terminal residue.

When in monomeric form, the above at least one disulfide bond is anintramolecular cystine disulfide formed between the at least two Cysresidues present in the antigenic polypeptide. When the antigenicpolypeptide is in a multimeric form that contains a plurality ofpolypeptide repeating units, the above at least one disulfide bond maybe an intramolecular cystine disulfide formed between the at least twoCys residues present in each polypeptide repeating unit, or thatdisulfide bond may be an intramolecular cystine disulfide formed betweenone of the at least two Cys residues present in a first repeating unitand another one of the at least two Cys residues present in a secondrepeating unit. Thus, whether in monomeric or multimeric forms, thecystine disulfide bond is intramolecular. However, in the monomeric ST,the disulfide bond is an intrapolypeptide bond, while in multimeric STembodiments the disulfide bond may be an intrapolypeptide orinterpolypeptide bond.

The above antigenic polypeptide may be used in its monomeric form aloneas when used in a diagnostic where competitive binding determinationsare carried out, or more preferably as a monomeric immunogen of avaccine when conjugated to a carrier molecule such as the porcineimmunoglobulin G. In still more preferred practice, the antigenicpolypeptide is utilized in a multimeric form.

When utilized in multimeric form, the polypeptide is one of a pluralityof repeating units of a multimer. In one embodiment, the multimercontains at least two of the antigenic ST polypeptides bonded togetherhead-to-tail through an amide bond formed between the amine group of theamino-terminus of one polypeptide and the carboxyl group of thecarboxy-terminus of the second polypeptide. In another multimericembodiment, the antigenic polypeptide is one of a plurality of repeatingunits of a polymer whose polypeptide repeating units are bonded togetherby interpolypeptide cystine disulfide bonds formed between the Cysresidues of the polypeptide repeating units.

In more preferred practice for the monomeric and multimeric forms ofsynthetic ST, with reference to the above formula for the antigenicsynthetic polypeptide:

a-f are integers having a value of zero or one with the proviso that:

"e" is zero when "a" is zero,

"d" is zero when "b" is zero, and

"f" is zero when "c" is zero;

the further proviso that at least one of "a", "b" or "c" must be zero sothat the corresponding R_(a-c) ¹⁻³ is absent as is the R_(d-f) ⁴⁻⁶ whosesubscript is zero when said "a", "b" or "c" is zero and anintramolecular cystine disulfide bond is present between the respectiveCys residues for which a subscript value of zero requires anothersubscript value to be zero; and

when a value of a-f is one; said R_(a-f) ¹⁻⁶ -groups, when takenindividually, are the same or different moieties bonded to the sulfuratom of the Cys residue and are selected from the group consistinghydrogen, an alkyl group containing 1 to about 4 carbon atoms and asubstituted alkyl group containing 2 to about 20 carbon atoms;

wherein g-l are integers having the value of zero or one, as notedabove, with the proviso that:

each of "g" and "k" is zero when "a" is zero,

each of "h" and "j" is zero when "b" is zero, and

each of "i" and "l" is zero when "c" is zero; and

when the value of g-l is one, the R_(g-l) ⁷⁻¹² -groups are the same ordifferent alternative amino acid residues to each immediately precedingCys residue.

The monomeric or multimeric antigenic synthetic polypeptide can thus beseen to contain at least one intramolecular disulfide that is anintrapolypeptide or interpolypeptide disulfide bond of a cystine residueformed between two Cys residues. In the more preferred embodiments, theintrapolypeptide cystine disulfide bond is formed between the pairs ofCys residues of groups R_(a) ¹ and R_(e) ⁵, or R_(b) ² and R_(d) ⁴, orR_(c) ³ and R_(f) ⁶. In still more preferred embodiments, the monomericsynthetic polypeptide contains at least two cystine residues and theirdisulfide bonds are formed betweenn the above pairs of Cys residues, andin most preferred embodiments, the synthetic polypeptide contains threecystine residues between the aforementioned Cys residues.

Biologic, natural (native) ST contains three disulfide bonds formedamong the six Cys residues. The most preferred monomeric synthetic ST ofthis invention has an identical 18-amino acid residue sequence to abiologic, native ST, and also contains three intramolecular,intrapolypeptide disulfide bonds formed among its six Cys residues.However, the thin layer chromatographic and electrophoretic mobilitiescompared with literature values, and immunologic properties of biologicand synthetic, monomeric ST molecules are different even though the twomolecules have the same primary structure and each contains threeintramolecular, intrapolypeptide disulfide bonds.

The monomeric or multimeric synthetic ST of this invention having atleast 10% of the antigenicity of biologic ST may be prepared bysynthesizing under non-oxidizing conditions a first, unoxidizedpolypeptide such as the polypeptide described before wherein hydrogen isthe R_(a-f) ¹⁻⁶ -group of the at least two of a-f that are one. Morepreferably, for the monomeric synthetic ST and the multimeric forms ofsynthetic ST, the first unoxidized polypeptide includes the amino acidresidue sequence taken from left to right and in the direction fromamino-terminus to carboxy-terminus represented by the formula:

    Cys(R.sub.g.sup.7)Cys(R.sub.h.sup.8)GluLeuCys(R.sub.i.sup.9)Cys(R.sub.j.sup.10) Tyr(Asn)ProAlaCys(R.sub.k.sup.11)Ala(Thr)GlyCysR.sub.l.sup.12 Asn(Tyr)

wherein the above more preferred amino acid residue sequence without thethree specific parenthesized amino acid residues and the R_(g-l) ⁷⁻¹²-groups corresponds to the amino acid residues of the ST Ib polypeptidenumbered 5 through 18 from the amino-terminus of that ST Ib polypeptide;

the three specific amino acid residues in parentheses are each analternative to the immediately preceeding amino acid residue;

R_(g) ⁷, R_(h) ⁸, R_(i) ⁹, R_(j) ¹⁰, R_(k) ¹¹ and R_(l) ¹² are the sameor different alternative amino acid residues to the preceding Cysresidue; g-1 are integers each having the value of zero or one with theproviso that if any of g-l has a value of zero the corresponding,individual R_(g-l) ⁷⁻¹² -group is absent, and the value of at least twoof g-l are zero, with the further proviso that at least one pair ofnon-contiguous Cys residues from the Cys residues preceding theindividual R_(g-l) ⁷⁻¹² -groups is present, the non-contiguous pairs ofCys residues being selected from the group consisting of the Cysresidues corresponding to amino acid residue positions in the ST Ibpolypeptide numbered 5 or 6 and 9 or 10, 5 or 6 and 14, and 9 or 10 and17 from the amino-terminus of the ST Ib polypeptide.

Once the first polypeptide is synthesized it is provided, and isdissolved or suspended in an aqueous composition at a concentration ofless than about 5 milligrams per milliliter, and more preferably at lessthan about 2 milligrams per milliliter, and most preferably at aconcentration of about 1 milligram per milliliter to about 0.1milligrams per milliliter. Preferably, the aqueous composition isalkaline and has a pH value less than about 10.5.

The obtained first polypeptide-containing composition is thereaftercontacted with molecular oxygen as an oxidizing agent. The contactbetween the composition and molecular oxygen is maintained for a periodof about 1 to about 24 hours to form at least one intrapolypeptide orone interpolypeptide cystine disulfide bond between the at least two Cysresidues present.

In preferred practice for the monomeric molecule, the disulfide bond isformed between the pairs of Cys residues corresponding to the amino acidresidues positions in the ST Ib polypeptide numbered 5 and 14, 6 and 10,and 9 and 17 from the amino-terminus of the ST Ib polypeptide. In morepreferred practice for that molecule, the contact between molecularoxygen and the solution containing the first polypeptide is maintainedfor a period sufficient to form two disulfide bonds between the abovementioned pairs of Cys residues, and still more preferably to formthree, intramolecular cystine disulfide bonds between those pairs of Cysresidues.

The present invention has several benefits and advantages. One suchbenefit is that it provides a source of immunologically active ST whichmay be prepared in large quantities and in substantially pure form. Oneof the advantages of the present invention is that the synthetic ST maybe used to vaccinate humans and other animals against strains of E. colithat produce ST. Another benefit of the present invention is that thesynthetic ST may be utilized in a diagnostic reagent or reagent systemfor assaying the presence of an infection caused by ST-producing E.coli. Yet another benefit of this invention is that ST molecules havingseveral times the antigenicity of the native toxin can be prepared.Still another advantage of the invention is that synthetic STpolypeptides can be prepared that have substantially less biologicactivity than the native toxin. Still further benefits and advantages ofthe present invention will be apparent to those skilled in the art fromthe detailed description examples and claims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming the portion of this disclosure:

FIG. 1 illustrates the potency of synthetic and biologic ST in inducingfluid secretion in the suckling mouse assay. One mouse unit (MU) isdefined as that amount of toxin which yields an intestinal (gut)weight:carcass weight ratio of at least 0.083. Values of MU shown areper microgram of toxin. The abscissa shows toxin dosage in nanograms(ng).

FIG. 2 illustrates fluid secretion following instillation of the toxinsfor 18 hours into ligated ileal loops of fasting rats. Values of theordinate are in microliters of fluid per centimeter of intestinallength. Values of the abscissa are in nanograms of toxin dosage. TheED₅₀ signifies that dosage in nanograms which evokes one-half of themaximum secretory response.

FIG. 3 illustrates the antigenicity of synthetic and biologic STdetermined by a double sandwich ELISA technique using hyperimmuneantisera to biologic ST. The antigenicity of the two ST toxins wassubstantially the same when tested against hyperimmune antisera tosynthetic ST. The ordinate is in units of optical density, while theabscissa is in nanograms of ST as antigen.

FIG. 4 illustrates the neutralizing effect of hyperimmune antisera toeither synthetic or biologic ST on the secretory effect of toxins in thesuckling mouse assay.

FIG. 5 shows the composition and properties of conjugates derived fromconjugating an initial molar ratio of synthetic ST to the LT holotoxinof 100:1 in the presence of varying concentrations of the carbodiimidereagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). Valuesfor the percent of ST in the final conjugates (ordinate) are based onweight of Lowry protein [Lowry et al., J. Biol. Chem., 193: 265-275(1951)]. The second, inset, abscissa shows residual LT holotoxintoxicity in the conjugate as a percentage of the toxicity ofnon-conjugated LT holotoxin.

FIG. 6 illustrates the effect of the initial molar ratio of synthetic STto LT B subunit on the amount of ST incorporated into the finalconjugate as determined using a tracer dose of radioiodinated ST. TheEDAC to conjugate ratio was 2:1.

FIG. 7 illustrates the effect of varying the EDAC to conjugate ratio onthe composition and antigenicity of conjugates obtained from an initialmolar ratio of synthetic ST to B subunit of 50:1. Data for ST are shownin the left panel, while data for the B subunit are shown in the rightpanel. Antigen units, expressed per 100 micrograms of conjugate, werederived by multiplying the percentage of toxin present (by weight) timesthe percentage of antigenicity. Circled numbers indicate the ratio ofEDAC to each specific toxin.

FIG. 8 illustrates the effect on composition and antigenicity of theconjugates of varying the initial molar ratio of synthetic ST to Bsubunit. The data are expressed as per FIG. 7. Circled numbers indicatethe ratio of EDAC to each specific toxin. The EDAC to conjugate ratiowas 1.5:1.

FIG. 9 illustrates protection attained in rats immunized with gradedantigen unit dosages of either the B subunit or synthetic ST coupled toporcine immunoglobulin G (PIG). Rats given varying dosages forintraparenteral (i.p.) primary immunization (left panel) all received atotal peroral (p.o.) booster dosage of 2,000 antigen units; those givenvariable p.o. boosters (right panel) all received i.p. primaryimmunization with 200 antigen units. Mucosal IgA antitoxin (mucosal AT)values in each panel are the increase in the reciprocal of the geometricmean titer in immunized rats over that of unimmunized controls. Numberswithin squares designate the ST mucosal AT values while numbers withincircles designate the mucosal AT values for the LT B subunit.

FIG. 10 illustrates protection attained against challenge with the humanLT⁺ /ST⁺ E. coli strain ( - - - ) compared to that against human LT- orST-only E. coli strains ( ---- ) in rats immunized with graded p.o.antigen unit dosages of either the B subunit or synthetic ST coupled toPIG. All rats received i.p. primary immunization with 200 antigen units.

FIG. 11 illustrates the effect of varying the initial molar ratio of ST( ---- ) to the B subunit ( - - - ) on the antigenicity of the componenttoxins in the cross-linked vaccine. The ratio of carbodiimide toconjugate protein was consistently 1.5:1 by weight.

FIG. 12 illustrates the immunogenicity of ST when given coupled to PIGor as a component of the cross-linked vaccine. All rats received (i.p.)primary immunization with 200 ST antigen units, followed by gradedperoral (po) boosts of ST as shown on the abscissa, and were thenchallenged with the human LT⁻ /ST⁺ E. coli strain. Mucosal AT values areshown by numbers within circles or squares and are as in FIG. 9.

FIG. 13 illustrates the toxicity of uncoupled, synthetic ST (left panel)and of that ST coupled to PIG as a vaccine (right panel) in the sucklingmouse assay. Values are the mean ± standard error of the mean for 3 micefor each datum point. MED, minimum effective dosage, is that dosage(here, in nanograms or micrograms) that yields a positive response of agut:carcass weight ratio of at least 0.083. Abscissas show the dosagesadministered in nanograms (left panel) or micrograms (right panel).

FIG. 14 illustrates the toxicity of uncoupled synthetic ST (left panel)and of that ST coupled to PIG as a vaccine (right panel) in rat ligatedileal loops. Values are the mean ± standard error of the mean for 3 ratsfor each datum point. ED₅₀ is that dosage in nanograms or microgramswhich yields one-half of the maximum secretory response. "Heated"indicates the vaccine was exposed to 65° C. for 1 hour prior to testing.Abscissas show the dosages administered in nanograms (left panel) ormicrograms (right panel).

FIG. 15 illustrates protection attained in immunized rats. Thehorizontal line designates 50% of maximum secretion in unimmunizedanimals, and ED₅₀ signifies that dosage in nanograms which produced thisvalue in this group of rats. PI signifies protection index, and IP/PO(intraperitoneal/peroral) signifies the immunization route. The upperpanel shows results from challenge with LT, while the lower panel showsresults from challenge with monomeric ST. The abscissa for both panelsshows the dosage of challenging toxin in nanograms. The ordinate showsthe volume of secretion in milliliters/centimeter of illeal loop.

FIG. 16 illustrates protection attained in immunized rabbits.Designations are the same as in FIG. 15, except that the challengingdosages and ED₅₀ values are in micrograms, and IM/PO signifiesimmunization by an intramuscular/peroral route.

FIG. 17 is a graph that illustrates the separation of a polymeric ST(P-ST) preparation using Sephadex G-50 column chromatography. Thenumerals of the abscissa refer to column eluate collection tube numbers,each tube receiving 4.5 milliliters of eluate, while the numerals of theordinate refer to the optical density of the collection tube contentsmeasured at 278 nanometers. The contents of collection tubes numbered5-9 were pooled to provide a solution containing substantially P-ST,while the contents of collection tubes numbered 10-18 were pooled toprovide a solution that primarily contained synthetic monomeric ST(M-ST).

FIG. 18 illustrates graphical representations of dose-secretory responsevalues for immunized ( ) and unimmunized ( ) rats that were challengedby instillation of graded dosages into ligated ileal loops for 18 hourswith E. coli ST (----) or with Klebsiella pneumoniae ST ( - - - ). Therats were immunized with a vaccine containing synthetic monomeric STconjugated to a natural LT B subunit as the immunogen. The abscissashows nanograms of the challenging toxin, while the ordinate shows thesecretory responses of those illeal loops in microliters per centimeterof loop. ED₅₀ signifies the dosage that produces 50 percent maximumsecretion in unimmunized rats. PI is the protection index obtained bydividing the dosage of toxin in immunized animals that yielded the samesecretion as the 50% effective dose (ED₅₀) in unimmunized animals by thevalue for unimmunized animals.

FIG. 19 is a graph that illustrates relative antigenicities of syntheticmonomeric ST (M-St; - - - ), a synthetic multimeric head-to-tail STdimer (ST/ST; - - - ) and of a preparation of polymeric synthetic ST(P-ST; Δ--.--Δ) as determined by a double-sandwich ELISA technique withhyperimmune antisera raised to an M-ST conjugate. Percent antigenicityis based upon the micrograms of ST required to yield an optical densityat 410 nanometers of 0.600. The abscissa shows the amount of ST permicrotiter plate well in micrograms.

FIG. 20 provides graphical illustrations of properties of threepreparations of ST conjugated to the LT B subunit using glutaraldehydeas coupling agent at a molar ratio of glutaraldehyde to the B subunit of700:1. Panel A illustrates conjugates prepared using synthetic monomericST (M-ST), panel B illustrates conjugates prepared using syntheticmultimeric head-to-tail ST dimer (ST/ST), and panel C illustratesconjugates prepared using polymeric synthetic ST (P-ST). The numerals ofthe abscissas of each of the panels represent the molar ratios of theappropriate ST preparation to B subunit in the initial conjugationreaction mixture. The left-hand ordinate for each of the panelsrepresents the percent of toxin by weight in the reaction mixtures, anddata points connected by solid lines (----) show molar ratios of the twotoxins in the reaction mixture. The right-hand ordinate for each panelshows the determined number of antigen units per milligram of vaccineprepared, and data points connected by dashed lines ( - - - ) show theantigenicity for each conjugate. Plots relating to the ST toxin areindicated by solid circles ( ), while plots relating to the B subunittoxin are indicated by open circles ( ). The letters "B" and "ST",designating data relating to the B subunit and to synthetic ST,respectively, are also placed adjacent each line for added clarity.Antigen units for the conjugates were determined by the double sandwichELISA technique with antisera to M-ST; ST values for all conjugates areexpressed in terms of M-ST antigen units.

FIG. 21 illustrates the immunogenicity of vaccines containing conjugatesof synthetic monomeric ST (M-ST) or of polymeric synthetic ST (P-ST)with the LT B subunit is providing reduced secretion (ordinate for bothpanels) and in increased mucosal IgA antitoxin titers afterimmunization. All rats received primary interperitoneal immunizationscontaining 200 antigen units of either conjugate followed by peroral(p.o.) boosts of differing amounts of the same vaccine. The rats werechallenged with a viable human LT⁻ /ST⁺ strain by instillation of thatstrain. The values for mucosal IgA antitoxin (AT) are circled and areshown as the mean increase in titers in immunized rats over those inunimmunized controls; AT in rats immunized with the vaccine containingthe P-ST conjugate was measured and is expressed as M-ST titers. Thedata of panel A are expressed as total peroral (p.o.) dosages in M-STantigen units, while the data in panel B are expressed as total peroral(p.o.) dosages in micrograms of ST, as M-ST, contained in theadministered, boosts.

FIG. 22 contains two panels of graphs that illustrate the percentage oftoxin by weight and the number of antigen units per milligram ofconjugates prepared from polymeric ST (P-ST) and the LT B subunit as afunction of the molar ratio of P-ST to B subunit of the conjugates.Properties of conjugates prepared using dimethyl suberimidate (DMS) areshown in panel A, while properties of conjugates prepared using EDAC[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] are shown in panel B.Data relating to the LT B subnit in both panels are shown as triangles,while data relating to P-ST are shown as circles. Filled in trianglesand circles ( , ), and solid lines refer to toxin percentage data, whileopen triangles and circles (Δ, ) and dashed lines refer to antigen unitdata. The ratios by weight of coupling agent to B subunit were 1:1 forDMS and 1.5:1 for EDAC.

DETAILED DESCRIPTION OF THE INVENTION I. Synthetic ST

The present invention contemplates synthetically produced polypeptideshaving the amino acid residue sequence of at least the carboxy-terminalfourteen amino acid residues of the heat-stable enterotoxin (ST)produced by Eschericia coli (E. coli). The synthetic polypeptides ofthis invention in monomeric and multimeric forms are useful asimmunogens or portions thereof of vaccines that can be used inprotecting against diarrheal infections produced by E. coli producingsuch toxins as well as against infections produced by other bacteriasuch as Klebsiella pneumoniae that produce similar toxins. The monomericand multimeric synthetic ST polypeptides and antibodies raised to themare also useful in diagnostics for assaying for the presence ofST-producing organisms, such as E. coli.

When used as a vaccine for immunizations, the synthetic ST polypeptidesmay be used alone, as is the case for the polymeric ST (P-ST) multimer,or used linked to a carrier as a conjugate as in the case for themonomeric ST (M-ST) and ST multimers. The synthetic ST immunogen ispresent in an effective amount in such a vaccine, and is dispersed ordissolved in a physiologically tolerable diluent.

Particularly useful conjugate carriers include the heat-labileenterotoxin of E. coli (LT) as well as the smaller, B subunit of thattoxin (LT B). Additionally useful carrier include porcine immunoglobulinG (PIG), keyhole limpet hemocyanin (KLH), tetanus toxoid,poly-L-(Lys:Glu), peanut agglutinin, olvalbumin, soybean agglutinin,bovine serum albumin (BSA), human serum albumin, and the like.

Physiologically tolerable (acceptable) diluents include water, saline,phosphate buffered saline (PBS), and the like, and typically furtherinclude an adjuvant. Complete Freund's adjuvant (CFA), incompleteFreund's adjuvant (IFA) and alum are typically used adjuvants that arewell known in the art, and are available commercially from severalsources.

The ST immunogen contained in a vaccine is present in an "effectiveamount", which amount depends upon a number of factors as is well knownin the immunological arts. Included among those factors are the bodyweight and species of animal to be immunized, the carrier when used andthe agent utilized to couple the ST to the carrier, the adjuvant whenused, the duration of protection desired, and the immunization protocolbeing utilized.

For example, in some challenge studies rats received about 1500 to about2500 antigen units (defined in Section III B, hereinafter) whenimmunized with an ST conjugate in a protocol wherein one intraparenteralinjection was followed by two peroral boosts. In another challengestudy, rabbits received about 2500 to about 3000 antigen units of anST-containing conjugate and were protected.

The weight of synthetic ST in such vaccines depends upon theantigenicities of the ST preparation utilized and of the ST-containingconjugate. Thus, using the knowledge that rats were protected fromchallenge when immunized with a total of about 1500 to about 2500antigen units of ST-containing vaccine, about 1500 to about 2500micrograms of an M-ST--LT B conjugate per rat was administered, whileabout 100 to about 300 micrograms of P-ST--LT B conjugate per rat wereadministered to achieve a similar result. A total of about 600micrograms of uncoupled (non-conjugated) P-ST were utilized in rabbitsto provide an antiserum titer sufficient to indicate protection fromchallenge.

It is thus seen that the amount of ST needed to provide an "effectiveamount" can vary widely. However, one skilled in the art can obtain thateffective amount using routine laboratory procedures from the discussionand citations that follow.

Synthetic ST embodying the present invention exhibits antigenicity, as amonomer alone or bound to a protein carrier as a conjugate or as amultimer alone or bound to a carrier as a conjugate, that is at leastabout 10 percent of that exhibited by biologic, native ST obtained fromE. coli which infect humans and other animals. It is known that themonomeric naturally occurring, biologic ST contains six Cys residueswhich form three intramolecular, intrapolypeptide disulfide bonds ofcystine residues. The presence of cystine disulfide bonds has been shownby others, Chan et al., supra., to be necessary for the functioning ofnaturally occurring ST.

Two of the six Cys residues of ST may theoretically combine in fifteendifferent ways to form a disulfide bond of one cystine residue. Sixpossible combinations of paired Cys residues remain for the formation ofa second disulfide bond, and thereafter only one combination is left forthe third disulfide bond. Thus, there are a total of ninety (15×6×1)theoretically possible secondary structural isomers of each primaryamino acid residue sequence of ST that contain six Cys residues and twoor three disulfide bonds.

However, because of the practical difficulty of forming a cystinedisulfide bond between pairs of contiguous Cys residues of which thereare two pairs in the ST molecule, and redundant possible structures,there are considerably fewer than ninety secondary structural isomerscontaining two or three disulfide bonds. The specific pairs of Cysresidues which form the disulfide bonds present in naturally occurringbiologic ST are not known.

It has now been found that an immunologically active synthetic ST can beprepared having a primary amino acid residue sequence that issubstantially the same as that of naturally occurring biologic ST, butwhose secondary structure, as determined by disulfide bond formation, isdifferent from that of naturally occurring ST. Antigenic activity isfound with monomeric synthetic ST molecules that contain but oneintrapolypeptide cystine disulfide bond among the pairs of Cys residuespresent. Enhanced antigenicity is provided to the synthetic monomeric STby formation of two disulfide bonds between two intrapolypeptide pairsof Cys residues, while maximum antigenic activity is provided by theformation of three intrapolypeptide disulfide bonds between three pairsof Cys residues. For multimeric forms of synthetic ST, at least oneintramolecular, intrapolypeptide cystine disulfide bond or oneintramolecular, interpolypeptide cystine disulfide bond is present, asis discussed hereinafter.

The synthetic ST utilized herein is described as being in monomeric andmultimeric forms. Monomeric synthetic ST contains at least the fourteenamino acid residues (14-mer) shown below in Formula I, including thevarious R-groups described. Multimeric synthetic ST contains a pluralityof at least 14-mer synthetic ST repeating units. In one embodiment, thesynthetic ST repeating units of the multimer are bonded togetherheat-to-tail; i.e., the amino-terminal amine of one repeating unit isbonded by an amide bond to the carboxy-terminal carboxyl of a secondsynthetic ST repeating unit to form a multimer containing at least twoST repeating units.

ST multimers containing ST repeating units bonded together head-to-tailwill sometimes be referred to hereinafter as dimer, trimer, tetramer,and the like ST multimers to indicate the number of ST repeating unitscontained in the multimer. A dimer ST multimer will also sometimes beindicated by the shorthand formula ST/ST, a timer ST multimer by theshorthand formula ST/ST/ST, and the like, with each "ST" representing atleast the 14-mer polypeptide whose sequence is shown in Formula I.

Another embodiment of this invention is an ST multimer that is referredto herein as "polymeric synthetic ST", as a "synthetic ST polymer" or as"P-ST". In that embodiment, the individual ST polypeptide repeatingunits are bonded together by interpolypeptide cystine disulfide bondsformed between and among the Cys residues of the ST repeating units.Each of the ST repeating units of polymeric syntehtic ST also containsat least the fourteen amino acid residues illustrated in Formula I.

The multimeric forms of ST of this invention may also be described interms frequently used in polymer chemistry. Using such terminology, thehead-to-tail multimeric ST, whose repeating units having an ST aminoacid residue sequence are bonded together by an amide bond between thecarboxy-terminal carboxyl group of one repeating unit and theamino-terminal amine group of a second repeating unit may also bedescribed as a straight-chain ST oligopolymer (oligomer), or as a blockoligomer whose repeating blocks have the amino acid residue sequence ofan ST polypeptide. The beforedescribed polymeric ST whose repeatingunits having an ST amino acid residue sequence are bonded together byinterpolypeptide cystine disulfide bonds is believed to be describableas a cross-linked or network polymer whose plurality of repeating unitshave the amino acid residue sequence of an ST polypeptide and whosecross-links are supplied by the interpolypeptide cystine disulfidebonds.

While retaining the above terminology, the above-described multimerscontaining repeating units having substantially only one ST amino acidresidue sequence may be termed straight-chain ST homo-oligopolymers orcross-linked (network) homopolymers, respectively. The multimers mayalso contain repeating units having different amino acid residuesequences or lengths, in which case those multimers may be termedstraight-chain ST co-oligopolymers or cross-linked (network) copolymers.

It must be understood that the above-described multimeric forms ofsynthetic ST represent only two of many possible ST multimers. Forexample, when the multimer referred to as a head-to-tail multimer orstraight-chain homo-oligopolymer having two repeating units is preparedby the oxidation of a 36-residue first polypeptide having an amino acidresidue sequence corresponding to that of two ST polypeptides, somepolymeric ST having interpolypeptide cystine disulfide bonds (a networkhomopolymer) is also formed. The repeating units of that networkhomopolymer contain two of the 18-mer amino acid residue sequences of STin the repeating unit. A copolymer may be prepared by the oxidation of18-mer ST first polypeptides along with the above 36-mer multimeric STfirst polypeptides to provide a network or cross-linked material whoserepeating units have an ST polypeptide amino acid residue sequence.

The word "synthetic" is utilized herein to mean that the ST polypeptidemolecule or polypeptide repeating unit has been built up by chemicalmeans (chemically synthesized) rather than by a biological means(biologically synthesized) such as that of the naturally occurringST-producing E. coli or by genetic engineering techniques. The syntheticpolypeptides are therefore free from naturally occurring proteins andfragments thereof. The well known solid phase chemical synthesis inwhich blocked amino acid residues are added in a serial manner toprovide a polypeptide is the preferred method of synthesis, and isdiscussed hereinafter.

A synthetic polypeptide according to this invention includes the aminoacid sequence as a monomer, alone, or as a repeating unit in a multimer,taken from left to right and in the direction from amino-terminus tocarboxy-terminus, represented by the sequence of Formula I: ##STR2##wherein the three specific amino acid residues in parentheses are eachan alternative to the immediately preceding amino acid residue in thesequence;

a, b, c, d, e and f (a-f) and g, h, i, j, k and l (g-l) are integerseach having a value of zero or one, with the proviso that if the valueof any of a-f or g-l is zero, the corresponding R_(a) ¹, R_(b) ², R_(c)³, R_(d) ⁴, R_(e) ⁵ or R_(f) ⁶ (R_(a-f) ¹⁻⁶ -) group or R_(g) ⁷, R_(h)⁸, R_(i) ⁹, R_(j) ¹⁰, R_(k) ¹¹ or R_(l) ¹² (R_(g-l) ⁷⁻¹² -) group isabsent, and when an R_(a-f) ¹⁻⁶ -group is absent the sulfur atom of theCys residue having an absent R_(a-f) ¹⁻⁶ -group forms a cystinedisulfide bond, while if the value of any one of a-f or g-l is one, thecorresponding R_(a-f) ¹⁻⁶ - or R_(g-l) ⁷⁻¹² -group is present;

the R_(a-f) ¹⁻⁶ -groups when taken individually, are the same ordifferent moieties bonded to the sulfur atom of the Cys residue and areselected from the group consisting of hydrogen, an alkyl groupcontaining 1 to about 4 carbon atoms, a substituted alkyl groupcontaining 2 to about 20 carbon atoms, an acyl group containing 1 toabout 8 carbon atoms, and a substituted acyl group containing 2 to about10 carbon atoms;

R_(g-l) ⁷⁻¹² are the same or different alternative amino acid residuesto each immediately preceding Cys residue; and

at least two of a-f and two of g-l are zero and two Cys residues arepresent with the proviso that the synthetic polypeptide contains atleast one intramolecular cystine disulfide bond formed from the at leasttwo Cys residues present.

When in monomeric form, the above at least one disulfide bond is anintramolecular, intrapolypeptide cystine disulfide formed between the atleast two Cys residues present in the antigenic polypeptide. When theantigenic polypeptide is in a multimeric form that contains a pluralityof polypeptide repeating units, the above at least one disulfide bondmay be an intramolecular, intrapolypeptide cystine disulfide formedbetween the at least two Cys residues present in each polypeptiderepeating unit, or that disulfide bond may be an intramolecular,interpolypeptide cystine disulfide bond formed between one of the atleast two Cys residues present in a first repeating unit and another oneof the at least two Cys residues present in a second repeating unit. Itis therefore seen that an intramolecular cystine disulfide bond ispresent in both monomeric and multimeric forms of ST. In the monomericST, that cystine disulfide bond is an intrapolypeptide bond, while inmultimeric ST the disulfide may be an interpolypeptide or anintrapolypeptide bond.

In more preferred practice for the monomeric and multimeric forms ofsynthetic ST, with reference to the above antigenic syntheticpolypeptide of Formula I:

"e" is zero when "a" is zero,

"d" is zero when "b" is zero, and

"f" is zero when "c" is zero; and

each of "g" and "k" is zero when "a" is zero,

each of "h" and "j" is zero when "b" is zero, and

each of "i" and "l" is zero when "c" is zero.

The sequence shown in Formula I without the three specific alternativeamino acids and substituent and alternative R-groups corresponds to thecarboxy-terminal fourteen amino acid residue sequence of ST Ib and ishomologous to amino acid residues numbered 59-72 of ST Ia from theamino-terminus. The fourteen amino acid residues comprising amino acids59-72 of ST Ia differ from the sequence illustrated above in Formula Iwithout its alternative amino acids and R-groups at position 65 whereinan asparagine (Asn) residue replaces the tyrosine (Tyr) residue at theposition numbered 11 from the amino-terminus of ST Ib (residue position8 from the carboxy-terminus), and at position 72 (carboxy-terminus)wherein a tyrosine residue replaces the asparagine residue shown.

Thus, the Tyr residue to the immediate right of the forth Cys residuefrom the amino-terminus (Tyr-65) may be replaced by the Asn residue thatis parenthesized in the above formula. A similar replacement of a Tyrresidue for Asn residue may also occur at the carboxy-terminus, as isshown by the parenthesization of the final Tyr residue.

The analogous fourteen amino acid residue sequence from ST Ic is thesame as that of ST Ib except for position 69 in ST Ic wherein athreonine (Thr) residue replaces an alanine (Ala) residue of ST Ib. Thatreplacement is also illustrated in the above formula by theparenthesized Thr residue.

It is particularly preferred that at least one of the fouramino-terminal amino acid residues present in the sequence of theeighteen residue ST Ib molecule also be present in its naturalpositional sequence in the synthetic ST. It is still more preferred thatall four of those additional amino acids be present in synthetic ST inthe same, natural positional sequence that they are present in ST Ib.

The preferred four additional amino acids at amino-terminus of thesynthetic ST molecule correspond to amino acid numbers 55 through 58 ofST Ia and are identical to those four amino acids in ST Ib. Three of thefour amino acids of ST Ic differ from those of either ST Ia or ST Ib atpositions 55 through 58 of ST Ia. Thus, using the above parenthesizedalternative naming system, the 4-amino acid polypeptide (4-mer) at theamino-terminus of the synthetic ST of Formula I in a most preferredembodiment has a sequence, taken from left to right and in the directionfrom amino-terminus to carboxy-terminus, represented, as shown below inFormula II: ##STR3## wherein the parenthesized amino acid may replacethe immediately preceding amino acid residue.

The monomeric synthetic polypeptide contains at least oneintramolecular, intrapolypeptide disulfide bond, more preferably twointramolecular, intrapolypeptide disulfide bonds and most preferablythree intramolecular, intrapolypeptide disulfide bonds. The disulfidebonds are believed to be formed between the pairs of Cys residues ofR_(a) ¹ and R_(e) ⁵, and R_(b) ² and R_(d) ⁴ as well as between the Cysresidues of R_(c) ³ and R_(f) ⁶, when a-f have the value of zero.

However, the Cys residues of R_(a) ¹ and R_(b) ² as well as those ofR_(c) ³ and R_(d) ⁴ are adjacent, contiguous pairs. Consequently,synthetic ST polypeptides containing one disulfide bond can havesubstantially similar secondary structures and antigenicities regardlessof whether that single disulfide bond is formed between the Cys residuesof R_(a) ¹ and R_(e) ⁵ or of R_(b) ² and R_(e) ⁵. Similar resultspertain to secondary structures formed due to disulfide formationbetween the Cys residues of R_(a) ¹ and R_(d) ⁴ rather than R_(b) ² andR_(f) ⁶, and the like.

The first polypeptide that is synthesized prior to the oxidativeformation of an intramolecular intrapolypeptide disulfide bond containsat least two Cys residues, so the value of at least two of g-l are zeroand the corresponding R_(g-l) ⁷⁻¹² groups are absent. In view of thesimilarity of secondary structur that is provided by formation of anintramolecular, intrapolypeptide cystine disulfide bond between one oftwo contiguous Cys residues and another Cys residue, a proviso is addedfor preferred monomeric and multimeric synthetic ST that at least onepair of non-contiguous Cys residues from the Cys residues preceding theR_(g-l) ⁷⁻¹² groups is present. That pair is selected from the groupconsisting of Cys residues that precede R_(g) ⁷, R_(h) ⁸ and R_(i) ⁹,R_(j) ¹⁰, R_(g) ⁷, R_(h) ⁸ and R_(k) ¹¹, and R_(i) ⁹, R_(j) ¹⁰ and R_(l)¹². In terms of the amino acid residue positions in ST Ib, the pairs ofnon-contiguous Cys residues in monomeric synthetic ST are selected fromthe group consisting of those numbered 5 or 6 and 9 or 10, 5 or 6 and14, and 9 or 10 and 17 from the amino-terminus of the ST Ib 18-merpolypeptide.

Multimeric synthetic ST molecules that are bonded together head-to-tailpreferably contain at least one intramolecular, intrapolypeptide cystinedisulfide bond per repeating unit. The intrapolypeptide cystinedisulfide bonds present in each repeating unit of such multimers arepreferably between the same Cys residues as those present in themonomeric molecules. The presence of two intrapolypeptide bonds betweenCys residues of each repeating unit is still more preferred, while thepresence of three intrapolypeptide cystine disulfide bonds per repeatingunit is mot preferred.

For the polymeric synthetic ST molecules wherein the polypeptide STrepeating units are bonded together by intramolecular, interpolypeptidecystine disulfide bonds, it is not believed necessary that the two Cysresidues present be non-contiguous. It is preferred, however, that thoseCys residues be non-contiguous as discussed above.

In addition to its primary amino acid residue sequence, a monomericsynthetic ST molecule containing one intrapolypeptide cystine disulfidebond among its six Cys residue is also conveniently characterized by itsantigenic activity being at least about 10 percent of the antigenicactivity of biologic ST containing three disulfide bonds. Differences inthin layer chromatographic and electrophoretic mobilities betweenmonomeric synthetic ST molecules containing three intramolecular,intrapolypeptide disulfide bonds and literature values for biologic STcan also be useful in characterizing synthetic ST molecules containingone intramolecular, intrapolypeptide disulfide bond when such asynthetic ST can form three intramolecular, intrapolypeptide cystinedisulfides.

Since similar pairings of Cys residues other than those that areparticularly preferred can also occur for monomeric synthetic STmolecules containing two intramolecular, intrapolypeptide disulfidebonds, such ST molecules are also conveniently characterized by havingan antigenic activity that is at least about 10 percent of that ofbiologic ST containing three disulfide bonds. Thin layer chromatographicand electrophoritic mobilities are again useful in characterizingmonomeric synthetic ST molecules containing two intramolecular,intrapolypeptide disulfide bonds when the third disulfide can also beformed.

The antigenicity of a synthetic ST as a percentage of the antigenicityof biologic ST is discussed in detail hereinafter in Section II.Broadly, however, the percentage of antigenicity of a synthetic ST is arelative measure of the amount of anti-biologic ST antibody thatrecognizes a synthetic ST compared to biologic ST recognized by the sameanti-biologic ST antibodies. Antigenicity calculations are based uponthe weight of ST antigen used, and are independent of whether theantigen assayed is monomeric or multimeric.

Suitable antigenicity has also been found for synthetic ST moleculeswherein the sulfur atoms of Cys residues comprise portions of linkagesother than cystine disulfide linkages. Because of that fact, the Cysresidues of the above sequence of Formula I for synthetic ST are shownas bonded to R_(a-f) ¹⁻⁶ groups whose identities are discussedhereinbelow.

It is noted however, that because at least one intramolecular,intrapolypeptide cystine disulfide bond is required for antigenicactivity in the monomeric sytnethic ST, and biological activity whenthat is desired, all six of the R_(a-f) ¹⁻⁶ groups may not be present inone synthetic ST molecule. Rather, only four of those groups may bepresent in any one molecule. Thus, for example, where the Cys residuesof R_(a) ¹ and R_(e) ⁵ are combined to form an intramolecular,intrapolypeptide cystine disulfide bond, the values of "a" and "e" arezero (below), the R_(a) ¹ - and R_(e) ⁵ -groups are absent and onlyR_(b) ², R_(c) ³, R_(d) ⁴, and R_(f) ⁶ may be present in a monomericsynthetic ST molecule.

To account for the presence of one, two or three intramoleculardisulfide bonds of cystine residues formed among the six Cys residues,each of the R-groups 1-6 has also been labeled with a subscript lettera-f. Each subscript letter represents an integer having a value of zeroor one. For more preferred embodiments, the proviso is added that "e" iszero when "a" is zero, "d" is zero when "b" is zero, and "f" is zerowhen "c" is zero, with the further proviso that at least one of "a", "b"or "c" must be zero, with the still further proviso that a disulfidebond is present between the respective pairs of Cys residues for whichone subscript value of zero requires another subscript value to also bezero.

Each of the R_(a-f) ¹⁻⁶ -groups present in the synthetic ST may behydrogen. In such a case, the Cys residue to which R_(a-f) ¹⁻⁶ -group isbonded is unsubstituted inasmuch as hydrogen is a normal group bonded tothe sulfur atom of a Cys residue. The presence of hydrogen bonded to thesulfur atom of a cysteine is denoted herein by the designations Cys orCysH.

The R_(a-f) ¹⁻⁶ -groups may also be alkyl groups that contain 1 to about4 carbon atoms. Exemplary of such R_(a-f) ¹⁻⁶ -groups are methyl, ethyl,propyl, i-propyl, n-butyl, i-butyl, and the like.

The R_(a-f) ¹⁻⁶ -groups may further be substituted alkyl groupscontaining 2 to about 20 carbon atoms wherein the substituents includearyl, substituted aryl, hydroxy, amino, carboxy, carboxamido, halo,substituted vinyl, substituted thio groups, and the like. Exemplary ofsuch substituted alkyl groups are benzyl, alpha-tolyl, 2-hydroxyethyl,2-hydroxypropyl, 2-aminoethyl, carboxymethyl (--CH₂ CO₂ H), carboxamidomethyl (--CH₂ CONH₂), p-chlorobenzyl, ethylene bis-acrylyl, 2-thioethanecarboxy (--SCH₂ CH₂ CO₂ H), and the like.

Included among the substituted alkyl groups from which the R_(a-f) ¹⁻⁶-groups may be selected is a linking group useful for bonding thesynthetic ST to another molecule such as an antigen carrier like the Bsubunit of an E. coli heat-labile (LT) toxin. Exemplary of such linkinggroups are diacrylates such as ethylene bis-acrylate which can react onedouble bond with a sulfur atom of a Cys group through a Michael additionreaction while leaving the second double bond free for bonding to acarrier; thioacetic acid, thiopropionic acid, cysteine andN-2-hydroxyethyl-2-thiosuccinimide which can form mixed disulfide bondswith a Cys residue sulfur atom and can react with another molecule suchas a carrier through their carboxyl groups, the cysteine amino group orthe hydroxyethyl group, respectively.

An R_(a-f) ¹⁻⁶ -group, in less preferred practice, can be an acyl groupcontaining 1 to about 8 carbon atoms, or a substituted acyl groupcontaining 2 to about 10 carbon atoms. Exemplary acyl groups containing1 to about 8 carbon atoms include formyl, acetyl, propionyl, hexanoyl,benzoyl and the like. Exemplary substituted acyl groups containing 2 toabout 10 carbon atoms include 2-methoxyacetyl, 3-ethoxypropionyl,4-chlorobenzoyl, 3-carboxypropionyl (a maleic anhydride adduct), and thelike. Acyl and substituted acyl groups are less preferred because theirthioester bonds are not particularly stable in aqueous media.

The R_(a-f) ¹⁻⁶ -groups may be present separately in a synthetic STmolecule, or mixtures of R_(a-f) ¹⁻⁶ -groups may be present in one STmolecule. When all of the subscript letters a-f of a monomeric syntheticST molecule have a value of zero, the R_(a-f) ¹⁻⁶ -groups are absent,and three intramolecular, intrapolypeptide cystine disulfide bonds arepresent.

The subscript letters a-f may also all have values of zero and theR_(a-f) ¹⁻⁶ -groups be absent in a polymerized synthetic ST molecule(P-ST) wherein intramolecular, interpolypeptide cystine disulfide bondsbetween synthetic ST polypeptide repeating units are present.Intramolecular, intrapolypeptide disulfide bonds within the synthetic STrepeating units may also be present in P-ST. On the average, therepeating units of such a polymer contain at least about two suchinterpolypeptide cystine bonds per repeating unit. Consequently, inpreferred practice at least two of a-f and two of g-l have a value ofzero for such polypeptide repeating units, and at least two R_(a-f) ¹⁻⁶-groups and two corresponding R_(g-l) ⁷⁻¹² -groups are absent due to theformation of the at least two interpolypeptide cystine disulfide bonds.

Antigenicity and biological activity can also be obtained usingsynthetic ST molecules containing at least one intramolecular,intrapolypeptide cystine disulfide bond between the pairs of Cysresidues such as those shown in Formula I as bonded to R_(a) ¹ and R_(e)⁵, R_(b) ² and R_(d) ⁴, or R_(c) ³ and R_(f) ⁶, corresponding to thepositions numbered 5 and 10, 6 and 14, and 9 and 17 from theamino-terminus of the ST Ib molecule, respectively, when the Cysresidues not included in the disulfide bond are replaced by the same ordifferent alternative amino acid residues. The preferred alternativeamino acid residues to the Cys residues of Formula I provide no ioniccharge to the synthetic polypeptide when the synthetic polypeptide isdissolved in an aqueous solution of physiological pH values; i.e., thepreferred alternative amino acid residues are free from ionic chargeswhen part of the polypeptide and in aqueous solution.

The alternative amino acid residues to the non-disulfide-bonding Cysgroups are illustrated in the above Formula I by the parenthesizedgroups R_(g) ⁷, R_(h) ⁸, R_(i) ⁹, R_(j) ¹⁰, R_(k) ¹¹ and R_(l) ¹² eachof which can replace the preceding Cys residue, and wherein thesubscripts g-l are integers having the value of zero or one. Inpreferred synthetic ST polypeptides, if "a" is zero, "g" and "k" areeach zero; if "b" is zero, "h" and "j" are each zero; and if "c" iszero, "i" and "l" are each zero.

The amino acid residues alanine (Ala) and serine (Ser) are exemplarly ofpreferred alternative amino acids that are useful for replacing Cysresidues. Biological and antigenic activities provided by synthetic STmolecules having Cys residues replaced by Ser residues are illustratedhereinafter.

The above disclosure as to groups R_(a) ¹ -R_(f) ⁶ and R_(g) ⁷ -R_(l) ¹²is equally applicable to synthetic ST polypeptides containing thefourteen amino acid residues shown in Formula I and to the morepreferred synthetic ST polypeptide containing 18-amino acid residueswhose sequence, taken from left to right and in the direction fromamino-terminus to carboxy-terminus, is represented by Formula III,below: ##STR4## wherein the parenthesized amino acid residues, and R_(a)¹ -R_(f) ⁶ and R_(g) ⁷ -R_(l) ¹² are as defined hereinbefore.

Both the preferred synthetic polypeptide of Formula I and the morepreferred synthetic polypeptide of Formula III may also include anadditional group R_(m) ¹³ bonded to the amine of amino-terminal residueof the polypeptide, wherein "m" is an integer having the value of zeroor one, with the proviso that if the value of "m" is zero, R_(m) ¹³ isabsent and the amino-terminal amine is unsubstituted. If the value of"m" is one, R_(m) ¹³ may be a peptide containing 1 to about 58 aminoacid residues, a linking group, and the acyl portion of a carboxylicacid containing 1 to about 20 carbon atoms forming an amide bond withthe amine of amino-terminal residue.

When R_(m) ¹³ includes additional amino acid residues bonded to theamino-terminus of the synthetic polypeptide of the fourteen residuepolypeptide of Formula I, the additional amino acid residues in onepreferred embodiment have the sequence, from the amino-terminus to thecarboxy-terminus, shown before in Formula II. Addition of the amino acidresidue sequence of Formula II to the preferred fourteen amino acidresidue sequence provides the sequence of the more preferred eighteenamino acid residue polypeptide of Formula III which itself correspondsto the carboxy-terminal amino acid residue sequences of ST Ia and ST Icand the entire ST Ib sequence written together as one sequence.

In preferred practice for the fourteen residue polypeptide, the value of"m" is one and R_(m) ¹³ is a chain containing the four amino acidresidue sequence of Formula II. In still more preferred practice, thealternative, parenthesized, amino acid residues of Formula II are absentand R_(m) ¹³ is a peptide chain containing the amino acid residuesequence of the four amino-terminal residues of ST Ib; i.e.AsnThrPheTyr, from left to right and in the direction fromamino-terminus to carboxy-terminus.

Bonding of 58 or 54 amino acid residues to the fourteen or eighteenresidue, synthetic ST polypeptides, respectively, can provide asynthetic polypeptide sequence corresponding to the sequence of nativeST Ia or ST Ic. However, since the preferred fourteen, and morepreferred eighteen, amino acid residue sequences of Formulas I and III,respectfully, can provide antigenic and biological activitiessubstantially the same as or better than those of the native ST,synthetic polypeptides containing amino acid sequences longer than aboutthe eighteen amino acid residues of Formula III so as to conform to theentire sequence of ST Ia and ST Ic are not necessary, nor are theydesired. However, additional amino acid residues may be usefully bondedto the amino-terminus of the polypeptides of Formulas I and III.

Thus, it can be desirable to include an additional Cys residue at thepolypeptide amino-terminus to use as a means of affixing the syntheticpolypeptide to another molecule, such as a carrier or a linking group.When used under such conditions, it is convenient to use a sulfur atomblocking group other than a conventional solid phase blocking group lestthe added amino-terminal Cys form a cystine disulfide bond with a Cysresidue internal to the sequence. One way that a Cys sulfur atom may beblocked during synthesis and selectively deblocked is through the use ofan isothiourea analogue of Cys which on cleavage results in theformation of a Cys residue.

In another preferred embodiment, a plurality of 14-mer or 18-mersynthetic ST polypeptides may be bonded together in a head-to-tailmanner to form one multimeric ST embodiment of this invention. Thismultimer contains at least two of the antigenic ST polypeptides andpreferably two to three of such repeating units, bonded together throughan amide bond formed between the amino group of the amino-terminus ofone polypeptide and the carboxyl group of the carboxy-terminus of thesecond polypeptide. An exemplary head-to-tail multimeric ST containingthirty-six amino acid residues, and having the amino acid residuesequence of two ST Ib polypeptides as repeating units is described indetail hereinafter.

Each of the additional ST repeating units can have the sequence shown inFormula I, hereinbefore. Multimeric synthetic ST molecules that containa plurality of synthetic ST repeating units bonded head-to-tail can beprepared by the solid phase synthetic method described hereinafter inSection II. Because of the difficulties involved with synthesizing largepolypeptide molecules, it is preferred that such head-to-tail multimerscontain about 2 to about 3 ST repeating units.

In still another preferred embodiment, the R_(m) ¹³ group may besynthetic polypeptide whose amino acid residue sequence corresponds to aprotein or polypeptide other than ST. For example, determinant aminoacid residue sequences from a toxin such as that of the E. coli LT Bsubunit may be utilized to provide a combined immunogen or antigenagainst both toxins. Useful amino acid residue sequences that correspondto determinants of the LT B subunit have been found to exist atpositions 37 through 62 [LT B-(37-62)] and at positions 27-36 [LTB-(27-36)] from the amino-terminus of the LT B whose complete amino acidresidue sequence was reported by Dallas et al., Nature, 288: 499-501(1982). Those amino acid residue sequences are shown below from left toright and in the direction of amino-terminus to carboxy-terminus:

LT B-(37-62) MetValIleIluThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLys, and

LT B-(27-36) TyrThrGluSerMetAlaGlyLysArgGlu.

The carboxy group of the carboxy-terminal residue of either of the abovepolypeptides may be bonded to the amine of the amino-terminal residue ofST by an amide bond therebetween. Such bonding provides another exampleof head-to-tail bonding as is useful in some multimeric ST embodiments,as described before. The LT B-(27-36) and LT B-(37-62) polypeptides mayalso be bonded together head-to-tail to provide an R_(m) ¹³ having theentire sequence from position 27 through 62 of the LT B subunit to theamino-terminus of ST.

It is particularly convenient to synthesize an unoxidized first STpolypeptide having the above LT B polypeptide bonded to theamino-terminal ST residue using the before discussed stepwise, solidphase technique. That first polypeptide is then oxidized to provide theamino-terminal, R_(m) ¹³ -group substituted ST. Such ST molecules willbe referred to hereinafter as LT B-(27-36)--ST or LT B-(37-62)--ST toindicate the presence of either of the LT Bsubunit-related polypeptidesas bonded to an ST.

Each of the above LT B-(27-36)--ST and LT B-(37-62)--ST polypeptides wascoupled separately to an equal weight of tetanus toxoid usingglutaraldehyde in a matter similar to that dicussed in Section VI Ahereinafter. Vaccines prepared from 400 micrograms of the purifiedconjugates was injected into separate rabbits in complete Freund'sadjuvant followed fourteen days later with boosts of the same amount ofimmunogen in incomplete Freund's adjuvant, and seven days thereafterwith boosts of the same amount of immunogen in alum. Seven daysthereafter (day 28), the rabbits were bled and antibodies to ST and tothe B subunit were collected. Titers of those antibodies against nativeB subunit and synthetic ST provided values of 10 and 160-320,respectively, for the LT B-(27-36)--ST immunogen, and values of 160 and1280, respectively, for the LT B-(37-62)--ST immunogen. Thus, theconjugates of both linked polypeptides are immunogenic, as well asantigenic (Table 2).

The R_(m) ¹³ group may also be linking group in addition to an added Cysresidue. Included among such linking groups are the reaction products ofthe synthetic polypeptide and: (i) omega-aminomonocarboxylic acidscontaining about 3 to about 6 carbon atoms such as beta-alanine and6-aminohexanoic acid, (ii) a dicarboxylic acid or acid chloride or acidanhydride containing about 3 to about 8 carbon atoms such as maleicacid, fumaric acid, succinic anhydride, phthalic anhydride, and thelike, (iii) a blocked mercaptan-containing carboxylic acid including 2to about 4 carbon atoms in the acid chain such as an isothiourea aderivative of thioglycolic or thiopropionic acids, (iv) a dialdehydecontaining about 2 to about 8 carbon atoms such as gluteraldehyde orp-phthaldehyde, and the like.

Thus, linking groups containing free amino, carboxyl, mercapto andaldehydo groups can be provided for use in bonding the syntheticpolypeptide to another molecule such as a carrier.

R_(m) ¹³ may also be the acyl portion of a monocarboxylic acidcontaining 1 to about 20 carbon atoms forming an amide bond with theamine of the amino-terminal residue of the synthetic polypeptide.Exemplarly of such monocarboxylic acid groups are acetic, propionic,hexanoic, lauric, myristic, stearic, oleic acids, and the like. A longchain fatty acid R_(m) ¹³ group such as stearoyl is usefully bonded to asynthetic ST for passive hemagglutination assays, while short chainedR_(m) ¹³ groups such as acetyl bonded to ST provide antigenic activitiesthat are slightly reduced compared to the underivatized eighteen residuepolypeptide.

Thus, the synthetic ST polypeptides of Formulas I and III containing theR_(m) ¹³ -group can be represented by the amino acid residue sequencesshown in Formulas I-A and III-A below, taken from left to right and inthe direction from amino-terminus to carboxy-terminus: ##STR5## whereineach specific amino acid residue in parentheses is an alternative to theimmediately preceding amino acid residue, and R_(a-f) ¹⁻⁶ -, R_(g-l)⁷⁻¹² - and R_(m) ¹³ -groups are as before described.

An embodiment of this invention containing the R_(m) ¹³ -group bonded tothe amino-terminal residue of an 18 residue synthetic ST that alsocontains two intramolecular, intrapolypeptide cystine disulfide bonds isrepresented, from left to right and in the direction from amino-terminusto carboxy-terminus, by the formula ##STR6## wherein the lines betweenCys residues represent intramolecular, intrapolypeptide cystinedisulfide bonds, each of the six specific amino acid residues inparentheses is an alternative to the immediately preceding amino acidresidue, and R_(a) ¹, R_(e) ⁵, R_(g) ⁷, R_(k) ¹¹, and R_(m) ¹³ are asbefore defined.

The most preferred 18 residue (18-mer) synthetic ST having threeintramolecular, intrapolypeptide cystine disulfide bonds is representedby the amino acid residue sequence shown in Formula IV below, taken fromleft to right in the direction from amino-terminus to carboxy-terminus:##STR7## wherein lines between Cys residues represent intramolecular,intrapolypeptide cystine disulfide bonds, and each of the six specificamino acid residues in parentheses is an alternative to the immediatelypreceding amino acid residue.

The above synthetic polypeptide of Formula IV represented as alsocontaining R_(m) ¹³ -group bonded to the amino-terminal residue is shownbelow, from left to right and in the direction from amino-terminus tocarboxy-terminus: ##STR8##

II. PREPARATION OF SYNTHETIC ST

The 18-residue human efflicting ST polypeptide (ST Ib) in unoxidizedform was synthesized by the well known solid phase method using aBeckman Model 990B Peptide Synthesizer, Beckman Instruments Co.,Berkeley, CA. See, Houghten et al., Int. J. Pept. Prot. Res., 16:311-320 (1980) and Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963),which disclosures are hereby incorporated by reference.

Briefly, for each synthesis, 1.00 gram (0.2-0.6 milliequivalents/gram)of benzhydryl amine resin was utilized with the initial protected aminoacid residue being alpha-O-benzyl-N-Boc-aspartic acid. Usual side-chainprotecting groups were used for the remaining amino acid residues asfollows: O-(p-bromobenzyoxycarbonyl) for tyrosine, O-benzyl forthreonine, serine and glutamic acid, and S-methoxybenzyl for cysteine.Protected amino acids were recrystallized from appropriate solvents togive single spots by thin layer chromatography. Couplings were typicallycarried out using a ten-fold molar excess of both protected amino acidand dicyclohexyl carbodiimide over the number of milliequivalents ofinitial N-terminal amino acid. A two molar excess of both reagents canalso be used. For asparagine, an equal molar amount ofN-hydroxy-benzotriazole was added to the protected amino acid anddimethylformamide was used as the solvent. All coupling reactions weremore than 99% complete by the picric acid test of Gisin, Anal. Chem.Act, 58: 248-249 (1972).

A portion of the resulting, protected polypeptide polymer (1 gram) wastreated with two milliliters of anisole, and anyhydrous hydrogenfluoride, 20 milliliters, was condensed into the reaction vessel at dryice temperature. The resulting mixture was stirred at 4° for 1.0 hour tocleave the protecting groups and remove the polypeptide from the resin.After evaporating the hydrogen fluoride at a temperature of 4° C. with astream of N₂, the residue was extracted with anhydrous diethyl etherthree times to remove the anisole, and the residue was dried in vacuo.

The vaccuum dried material was first extracted with 5% aqueous aceticacid (3 times 50 milliliters each) followed by extractions using 50%aqueous acetic acid (4 times 50 milliliters). The first extractionremoved low molecular weight polypeptides and tyrosine that was used insome preparations to protect the Cys mercapto groups. The secondextraction separated the free polypeptide from the resin. After dilutionwith water to a concentration of 10-20% acetic acid, the resultingsolution was lyophilized to provide a monomeric unoxidized, firstpolypeptide.

A typical 14-residue first monomeric polypeptide so produced isrepresented by the amino acid residue sequence, taken from left toright, and in the direction from amino-terminus to carboxy-terminus,shown in Formula V, below: ##STR9##

wherein each of the three specific amino acid residues in parentheses isan alternative to the immediately preceding amino acid residue, andR_(g-l) ⁷⁻¹² are as defined hereinbefore.

A typical 18-amino acid residue-containing synthetic first monomericpolypeptide so produced is represented by the amino acid residuesequence, taken from left to right in the direction from amino-terminusto carboxy-terminus, shown in Formula VI, below: ##STR10## wherein eachof the six specific amino acid residues in parentheses is an alternativeto the immediately preceding amino acid residue, and R_(g-l) ⁷⁻¹² are asdefined hereinabove.

A first, monomeric polypeptide whose amino acid sequence corresponds tothe sequences shown in Formulas I or III may also be prepared in whichat least two of a-f and g-l are zero, so that at least two Cys (CysH)residues are present as discussed previously. Such polypeptides may alsoinclude the previously discussed R_(m) ¹³ group.

The first, monomeric polypeptide can be utilized in the oxidation stepin the state of purity obtained after the above lyophilization or itspurity can be increased by passage through chromatographic columnscontaining Sephadex G-10 or G-50 resins (Pharmacia, Piscataway, N.J.)and/or DEAE-Sephacel (Pharmacia) following the method of Staples et al.,supra. In either case, it is important that the first, monomericpolypeptide so made be protected from premature oxidation. Consequently,manipulations on the first, monomeric polypeptide subsequent to itscleavage from the resin support and removal of protecting groups fromfunctional group-containing amino acid residues are carried out undernon-oxidizing conditions.

After oxidation of the Cys sulfhydryl groups to form the intramolecularcystine disulfide bonds, described hereinafter, there remained no freesulfhydryl groups. The presence or absence of free sulfhydryl groups,Cys mercapto groups, was determined by the method of Ellman, Arch.Biochem. Biophys., 82: 70-77 (1959).

The crude, oxidized monomeric polypeptide product was purified by gelfiltration on Sephadex G-10 (Pharmacia) followed by ion exchangechromatography on DEAE-Sephacel (Pharmacia). Amino acid analysis, geland paper electrophoresis and other chromatographic data all indicated ahomogenous product. The overall yield was approximately 25% of theory.Synthetic ST preparations prepared on 5 separate occasions were examinedfor biologic potency and antigenicity. All yielded substantially thesame responses described herein for the preparation described.

The polymeric ST multimers whose plurality of ST repeating units arebonded together by intramolecular, interpolypeptide cystine disulfidebonds were prepared from the monomeric polypeptide products preparedabove, following the oxidation procedure described hereinafter.

A first ST multimeric dimer (ST/ST) that is bonded together in ahead-to-tail manner, was prepared by the above solid phase method. Here,a first ST/ST multimer, each of whose repeating units had the ST Ib18-mer sequence, was prepared by serial addition of thirty-sixappropriately blocked amino acid residues, following the above procedurefor the 18-mer first ST polypeptide, and then repeating the samesequence for the next eighteen residues until the 36-mer firstpolypeptide was prepared.

OXIDATION PROCEDURE FOR MONOMERIC ST

A first polypeptide having the 18-residue sequence of ST Ib was preparedas discussed above, and had the amino acid residue sequence, taken fromleft to right in the direction from amino-terminus to carboxy-terminus,shown in Formula VII, below: ##STR11## This first polypeptide (50milligrams) was added with gentle agitation to an aqueous 0.1 molarammonium carbonate-containing solution (pH value 7.8-8.3) to provide afinal concentration of 1 milligram of deblocked first polypeptide permilliliter of solution. Continued gentle stirring over a period of 5-15minutes at room temperature provided a clear solution of the firstpolypeptide.

Gentle agitation at room temperature was continued for a total period of8 hours during which time molecular oxygen (O₂) in the air was contactedwith the solution as oxidizing agent to oxidize the six Cys residues andform three intramolecular, intrapolypeptide cystine disulfide bonds. Theloss of free sulfhydryl groups was followed with Ellman reagent [Ellman,Arch. Biochem. Biophys., 82: 70-77 (1959)], and was found to be 15%,40%, 60% and 98% complete at 0.5, 1.5, 2.5 and 8.0 hours, respectively.

The resulting, oxidized polypeptide was collected by lyophilization. Itcould be used in crude from, but was purified by column chromatography.

The crude material was first passed through a Sephadex G-50 (Pharmacia)column (1.5×80 centimeters) equilibrated with 0.1 molar ammoniumacetate. Fractions of 3.5 milliliters/20 minutes were collected withmaterials being found in fractions 12-15 and 22-28. The contents ofthese fractions were collected by lyophilization and provided 10 and 36milligrams, respectively. The material in fractions 22-28 was monomericsynthetic ST as indicated by its elution being in the identical positionfound by chromatography of biologic ST. Polymeric synthtic ST whosepreparation is discussed further hereinafter was also obtained by thisseparation.

The discussion hereinbelow relates to monomeric ST, and is followed by adiscussion of the preparation of multimeric synthetic ST molecules underthe sub-heading "Preparation of Multimeric ST".

The partially purified monomeric synthetic ST could again be used as is,but was purified still further by chromatography on a DEAE-Bio Gel A(Bio Rad, San Raphael, CA) column (1.0×25 centimeters). Elution wascarried out using a stepwise gradient with the principal amount ofmaterial eluting between 50 and 100 millimolar sodium chloride. Thatmaterial was collected by lyophilization.

The lyophilized material was redissolved in water and desalted on aSephadex G-25 (Pharmacia) column. The resulting material was collectedby lyophilization to yield 28 milligrams of pure, synthetic ST,representing a yield of 56 percent based upon the weight of the crude,first polypeptide. Amino acid analysis of the synthetic ST so preparedgave the following values based on an 18-residue polypeptide(theoretical in parentheses):

    ______________________________________                                        aspartic acid         1.93 (2.0)                                              threonine             1.98 (2.0)                                              glutamic acid         0.97 (1.0)                                              proline               1.05 (1.0)                                              glycine               1.00 (1.0)                                              alanine               2.00 (2.0)                                              leucine               1.05 (1.0)                                              tyrosine              1.89 (2.0)                                              phenylalanine         0.96 (1.0)                                              cysteine (as          5.80 (6.0)                                              cysteic acid)                                                                 ______________________________________                                    

The biological activity of this synthetic ST was determined by thesuckling mouse assay of Giannella, Infect. Immunity, 14: 95-99 (1976),and was found to be substantially the same as biologic ST as is shown inFIG. 1. Substantial identity of secretory responses in ligated ilealloops between synthetic and biologic ST molecules is shown in FIG. 2.

The more important antigenicity of synthetic ST was compared to that ofbiologic ST by reactivity to antibodies to biologic ST using the ELISAtechnique of Klipstein et al., Infect Immunol., 37: 550-557 (1982).These results are illustrated in FIG. 3 which shows that theantigenicity of the synthetic ST of this preparation was about 70% thatof biologic ST. However, FIG. 4 illustrates that seroneutralization onsecretory effects by hyperimmune sera were almost identical.

LOCATION OF INTRAMOLECULAR DISULFIDE BONDS IN MONOMERIC ST

The three intramolecular, intrapolypeptide disulfide bonds were found toform at different rates. This finding permitted identification of thelocation of the pairs of Cys residues which combine to form thedisulfide bonds.

Thus, further preparations of the above first polypeptide were oxidizedunder similar conditions and the free sulfhydryl groups were alkylatedwith iodoacetic acid or idoacetamide at various times during theoxidation reaction. The resulting partially oxidized-partially alkylatedpolypeptides were then sequenced using a Beckman Model 890 Sequencer(Beckman Instruments Co.) to determine which Cys residues were alkylatedat which times during the oxidation reaction. The ratio of alkylated Cysresidues at given positions in the partially alkylated-partiallyoxidized ST compared to the all alkylated-unoxidized ST reflected thelocation and order of formation of the disulfide bonds.

A ten-fold molar excess of alkylating agent was used over the moles ofCys residue. The oxidation-alkylation reaction mixture was stirred for aperiod of ten minutes subsequent to the addition of the alkylatingagent, followed by addition of a ten fold excess of dithiothreitol overalkylating agent and a further stirring period of one hour to consumethe alkylating agent.

The intramolecular, intrapolypeptide cystine disulfide bonds in themonomeric synthetic ST were found to be formed between the first andfifth, second and fourth, and third and sixth Cys residues from theamino-terminus; those Cys residues correspond to the residue of ST Ibnumbered 5 and 10, 6 and 14, and 9 and 17, respectively, from theamino-terminus. Those Cys residues also correspond to the Cys residuesbonded to R_(a) ¹ and R_(e) ⁵, R_(b) ² and R_(d) ⁴, and R_(c) ³ andR_(f) ⁶, respectively, whose positions from the carboxy-terminus in the18-residue polypeptide herein prepared are analogous to thecarboxy-terminal positions in the 14-residue polypeptide shown inFormula I.

The rate of formation was found to be in the order of the Cys residuesof R_(b) ² and R_(d) ⁴, followed by Cys residues of R_(c) ³ and R_(f) ⁶,and then followed by the Cys residues of R_(a) ¹ and R_(e) ⁵. Usingnumbering from the amino-terminus of ST Ib, the order of disulfide bondformation was between the Cys residues numbered 6 and 14, then 9 and 17,followed by 5 and 10.

The primary and secondary structure of the monomeric synthetic ST soprepared, was, from amino-terminus to carboxyl-terminus, therefore:##STR12## wherein the lines connecting the Cys residues represent theintramolecular, intrapolypeptide cystine disulfide bonds formed betweenthose residues.

The rate of intramolecular, intrapolypeptide cystine disulfide bondformation as a function of pH value using the above human ST firstpolypeptide and oxidation with molecular oxygen contacted with a gentlystirring solution containing 1 milligram per milliliter of the firstpolypeptide is shown below:

    ______________________________________                                                    Moles of --S--S--                                                 pH value    formed/hour                                                       ______________________________________                                        5.0         0.30                                                              6.0         0.45                                                              7.0         0.90                                                              8.0         1.50                                                              9.0         1.20                                                              10.0        0.75                                                              ______________________________________                                    

Stirring speed and temperature also effect the rate of oxidation.Increases in either or both provide a more rapid rate of oxidation anddisulfide bond formation.

A study was also carried out to assess the effect of the pH value atwhich the oxidation of the first polypeptide was conducted. Here, theabove 18-mer first polypeptide was agin utilized, and was oxidized atroom temperature, at a concentration of 1.0 milligrams per milliliter,and until no free sulfhydryl groups could be detected with Ellmanreagent, as discussed before. The first polypeptides were dissolved inan aqueous solution containing 0.1 molar ammonia. After dissolution, thepH values of the solutions were lowered using a solution of 50 percentaqueous acetic acid. Antigenicities after lyophilization and withoutpurification were determined relative to the antigenicity of biologic STusing the before-discussed ELISA technique. The results are shown below:

    ______________________________________                                                          Antigenicity                                                pH value          (% of biologic ST)                                          ______________________________________                                        8.5                75 (39)                                                    8.0                60 (32)                                                    7.8               300 (159)                                                   7.7               250 (132)                                                   7.6               150 (79)                                                    7.4                23 (12)                                                    7.2                46 (24)                                                    ______________________________________                                    

COMPARISON OF PHYSICAL PROPERTIES OF MONOMERIC SYNTHETIC AND BIOLOGIC ST

Thin layer chromatography using cellulose coated plastic sheets (EastmanKodak, Inc., Nutley, NJ) and a solvent of butanol:acetic acid:water(200:30:75 parts by volume, respectively) provided an R_(F) value of0.307 for the above prepared synthetic ST having three; intrapolypeptidedisulfide bonds. Staples et al., supra, reported an R_(F) value of0.8-0.9 using the same solvent system and cellulose coated glass plates(Eastman).

Paper electrophoresis at pH 2.1, 500 volts for 90 minutes at roomtemperature provided an R_(F) value of 0.80 relative to lysine for theabove synthetic ST. Staples et al., supra, using thin layerelectrophoresis on cellulose at pH 1.9 reported a mobility for biologicST that was about the same as that of glutamic acid. Conversion ofglutamic acid mobility under the Staples et al. condition to that oflysine under the conditions used for the synthetic ST provides arelative R_(F) of 0.50 for biologic ST.

Preliminary results from optical rotatory dispersion determinationsusing the above synthetic ST and its biologic counterpart showed thatthe two molecules are different.

MONOMERIC SYNTHETIC ST MOLECULES CONTAINING TWO DISULFIDE BONDS

Synthetic 18-residue polypeptides analogous to human ST Ib moleculeswere prepared in a manner substantially the same as that describedabove, except that pairs of Cys residues were replaced in the firstpolypeptide and resulting, oxidized, monomeric ST molecules byalternative R_(g-l) ⁷⁻¹² -groups which provide no ionic charge to thesynthetic ST polypeptide when that polypeptide is dissolved in aqueoussolution at physiological pH values. The synthetic ST molecules soprepared were then assayed for their antigenic and biologicalactivities. The R_(g-l) ⁷⁻¹² -groups used in these determinations werethe amino acid serine (Ser).

The first polypeptide from which these synthetic ST Ib analogues wereprepared had the amino acid sequence, taken from left to right in thedirection from amino-terminus to carboxy-terminus, shown in FormulaVIII, below: ##STR13## wherein each of the R_(g-l) ⁷⁻¹² -groups was analternative Ser residue to the immediately preceding Cys residue.

For this group of immunological assays as well as for all of the othersuch assays, the haptenic synthetic ST Ib and biologic ST Ib moleculeswere first coupled to porcine immunoglobulin G (PIG) as a carrier insubstnatially the same amounts and under substantially the sameconditions, as discussed in Section IV, hereinafter. Antisera were thenraised to the immunogens so produced. The abilities of those twoantisera to recognize each of the ST molecules were measured andcompared by the before-discussed ELISA technique of Klipstein et al.,Infect. Immun., 37: 550-557 (1982). Recognitions of purified syntheticST and biologic ST by their own antisera were set at 100% and theamounts of recognition for each of the other ST molecule was thencalculated accordingly. The suckling mouse assay was used fordetermining biological activity.

Thus, for these comparative determinations, pairs of R_(g-l) ⁷⁻¹² wereutilized in which the value of four of g-l was zero, while the value oftwo of g-l was one. The pairs of alternative R_(g-l) ⁷⁻¹² -groupsutilized were those whose preceding pairs of Cys residues are shown inFormula I for the 14-residue polypeptide to be bonded to the groupsR_(a) ¹ and R_(e) ⁵, R_(e) ³ and R_(f) ⁶, and R_(b) ² and R_(d) ⁴. Thesegroups correspond to the Cys residues numbered 5 and 10, 6 and 14, and 9and 17, respectively, from the amino-terminus of the ST Ib amino acidsequence. The R_(g-l) ⁷⁻¹² -groups for which g-l were one were thereforeR_(g) ⁷ and R_(k) ¹¹, R_(i) ⁹ and R₁ ¹², and R_(h) ⁸ and R_(j) ¹⁰,respectively, of the amino acid sequence of Formula VIII. The resultsfor these comparisons are shown in Table 1 below along with standards ofbiologic ST Ib, the above-prepared synthetic ST Ib and porcine ST Ia.

                  TABLE 1                                                         ______________________________________                                        Relative Antigenic and Biological Activities                                            Assay                                                                           Anti-    Anti-      Suckling                                      ST          Syn. ST.sup.1                                                                          Biol.-ST.sup.2                                                                           Mouse                                         Assayed     (Percent)                                                                              (Percent)  (Percent)                                     ______________________________________                                        R.sub.g.sup.7 + R.sub.k.sup.11                                                            45        62         66 (8.2 ng.sup.3)                            Ser                                                                           R.sub.i.sup.9 + R.sub.l.sup.12                                                            21        43         46 (12.0 ng.sup.3)                           Ser                                                                           R.sub.h.sup.8 + R.sub.j.sup.10                                                            41        74         92 (6.0 ng.sup.3)                            Ser                                                                           Biologic Ib 35       100        100 (5.7 ng.sup.3)                            Synthetic Ib                                                                              100      263        120 (5.3 ng.sup.3)                            Porcine Ia   3        450.sup.4  10 (55 ng.sup.3)                             ______________________________________                                         .sup.1 Percent recognition of assayed ST by antiserum raised to synthetic     ST.                                                                           .sup.2 Percent recognition of assayed ST by antiserum raised to biologic      ST.                                                                           .sup.3 Nanograms required to provide a gut: whole body ratio of 0.083.        .sup.4 This value appears to be anomolously high by a factor of about 1.5     to about 2.                                                              

The results in the above Table illustrate several features of thepresent invention. First, substantial immunologic activity, e.g.antigenicity, can be obtained without the presence of three disulfidebonds in the ST molecule. Second, and a related feature, all of the Cysresidues are not needed and some may be replaced by other amino acidresidues. Third, biological activity which can lead to diarrhea in avaccinated animal can be reduced while substantial antigenic activity ismaintained.

A fourth feature of this invention that the above results illustrate isthat the biologic ST Ib molecule and the synthetic ST Ib moleculecontaining three disulfide bonds are immunologically different entities,thereby underscoring the before-noted differences in chromatographic,electrophoretic and optical rotary dispersion chracteristics of the twomolecules. Thus, antibodies to biologic ST recognized the synthetic ST263% better than they recognized the biologic ST to which they wereraised. Similarly, antibodies raised to the synthetic ST recognizedbiologic ST only 35% as well as they recognized the synthetic ST.

The difference between the synthetic and natural ST molecules is furtherunderscored by the ST Ia (porcine) which was hardly recognized byantibodies to the synthetic ST, but was recognized about 10- to about100-times better by antibodies raised to biologic ST than was biologicST itself, taking into account the fact that the 450% value may beanomolously high. Even if the value of 450% is too high by a factor ofabout 5, the antibodies raised to biologic ST Ib recognized porcine STIa at least about 20-times better than did antibodies to the syntheticST Ib. If the biologic and synthetic ST Ib molecules were the same,antibodies to each would be expected to recognize the porcine ST Ia withabout the same efficiency. Since that was decidely not the case, thebiologic ST Ib and synthetic ST Ib molecules must be different althoughboth contain identical primary amino acid sequences and both containthree intramolecular disulfide bonds.

OTHER PREPARATIONS OF MONOMERIC ST

Additional first polypeptides and ST molecules have been prepared (i)via different oxidation routes, (ii) having different amino acidsequences from the ST Ib, (iii) replacements of different R_(g-l) ⁷⁻¹²-groups, (iv) alkylated Cys R_(a-f) ¹⁻⁶ -groups and (v) R_(m) ¹³ groupsbonded to the N-terminal amino acid residue of the 18-residue ST Ibmolecule. Many of these materials have been assayed for antigenic and/orbiological activities by the above ELISA and suckling mouse techniques,respectively using antisera to the synthetic ST prepared in (3), below.The results of several of these preparations and assays are listed belowin Table 2.

                  TABLE 2                                                         ______________________________________                                        Other Preparations                                                                                           Suckling.sup.3                                                       ELISA.sup.2                                                                            Mouse                                          Oxidations.sup.1      (Percent)                                                                              (Percent)                                      ______________________________________                                        (1) Room temperature; 1.0 mg/ml;                                                                    95       120 (5.3 ng)                                   0.1  --M NH.sub.4 HCO.sub.3 ; pH 8.0; 6 hrs.                                  (2) 4° C.; 1.0 mg/ml;                                                                        87        80 (6.9 ng)                                   0.1  --M NH.sub.4 HCO.sub.3 ; pH 8.0; 6 hrs.                                  (3) Room temperature; 1.0 mg/ml;                                                                    27        85 (6.5 ng)                                   0.1  --M NH.sub.3 ; pH 10.3; 1.0 hr.;                                         lyophilized; 4° C.; 1.0 mg/ml;                                         buffered saline; pH 7.2; 8 hrs.                                               (4) Room temperature; 1.0 mg/ml;                                                                    less     N.D..sup.4                                     0.1  --M NH.sub.3 ; pH 10.3; adjusted                                                               than 2                                                  with acetic acid to pH 8.0 after                                              5 minutes; 1 equivalent                                                       K.sub.3 Fe(CN).sub.6 ; 3 hrs.                                                 (5) Room temperature; 2.0 mg/ml;                                                                    13        55 (10 ng)                                    0.1  --M NH.sub.3 ; pH 10.3; 1 hr.;                                           lyophilized; 4° C.; 2.0 mg/ml;                                         buffered saline; 8 hrs.                                                       (6) Room temperature; 1.0 mg/ml;                                                                    much less                                                                              N.D.                                           1.0 mg/ml; performic acid                                                                           than 1                                                  (7) Room temperature; 1.0 mg/ml;                                                                    53       N.D.                                           0.1  --M NH.sub.3 ; pH 10.3; adjusted                                         immediately upon solution with                                                acetic acid to pH 8.0; 22 hrs.                                                (8) Room temperature; 1.0 mg/ml;                                                                    26       N.D.                                           0.1  --M NH.sub.3 ; pH 10.3; 22 hrs.                                          (9) Room temperature; 1.0 mg/ml;                                                                    100      N.D.                                           0.1  --M NH.sub.4 HCO.sub.3 ; pH 8.0; 8 hrs.;                                 Sephadex G-10 and DEAE-Bio Gel A;                                             lyophylize.                                                                   (10) Room temperature; 1 mg/ml;                                                                     7        N.D.                                           8  --M urea; pH 8.0; 8 hr.                                                    Alkylation                                                                    (11) Oxidize as per (1); alkylate                                                                   12       N.D.                                           with average of 4 moles iodoacetic                                            acid after an average of one                                                  disulfide formed.                                                             (12) Oxidize as per (1); alkylate                                                                   14       N.D.                                           with average of 2 moles of                                                    iodoacetic acid after an average of                                           two disulfides formed.                                                        Acyl ST (R.sub.m.sup.13)                                                      (13) An N--acetyl group was added                                                                   53       N.D.                                           to the amino-terminus of the 18-                                              amino acid residue of the first                                               polypeptide prior to removal of                                               the peptide blocking groups,                                                  followed by deblocking and                                                    oxidation as in (7).                                                          (14) LT B-(37-62)--ST;                                                                              147      N.D.                                           oxidized as per (1).                                                          (15) LT B-(27-36)--ST;                                                                              24       N.D.                                           room temperature; 0.1 mg/ml;                                                  0.5  --M NH.sub.4 HCO.sub.3 ; pH 8.0;                                         over night                                                                    (16) LT B-(27-36)--ST;                                                                              121      N.D.                                           room temperature; 1.0 mg/ml;                                                  0.5  --M NH.sub.4 HCO.sub.3 ; pH 8.0;                                         over night                                                                    (17) LT B-(27-36)--ST;                                                                              92       N.D.                                           room temperature; 5.0 mg/mlg                                                  0.5  --M NH.sub.4 HCO.sub.3 ; pH 8.0;                                         over night.                                                                   Alternative sequences                                                         (18) Carboxy-terminal 14 amino                                                                      6.sup.5  N.D.                                           acids of ST Ib, oxidized as per (9).                                          (19) Carboxy-terminal 15 amino                                                                      55       N.D.                                           acids of ST Ib, oxidized as per (9).                                          (20) Cys residues preceding                                                                         71       N.D.                                           Formula VIII R.sub.h.sup.8 and R.sub.i.sup.9                                  replaced by Ser, oxidized as per (9).                                         (21) Cys residues preceding                                                                         152      N.D.                                           Formula VIII R.sub.i.sup.9 and R.sub.k.sup.11                                 replaced by Ser, oxidized as per (9).                                         (22) Cys residues preceding                                                                         27       N.D.                                           Formula VIII R.sub.j.sup.10 and R.sub.k.sup.11                                replaced by Ser, oxidized as per (9).                                         ______________________________________                                         .sup.1 A first polypeptide corresponding to the sequence of Formula VIII      wherein g-1 were zero was used for each of (1)-(17). First polypeptides       corresponding to the ST Ib carboxyterminal 14amino acids and 15amino acid     were used in (18) and (19), respectively. First polypeptides correspondin     to the noted substitutions to the sequence in Formula VIII were used for      (20)-(22) and were then oxidized. Reaction conditions are provided in the     order of temperature; concentration of first polypeptide in                   milligrams/milliliter; molar concentration of added ingredients; pH value     at which oxidation was initiated; and duration of the oxidation procedure     Each solution was stirred gently to contact the solution with atmospheric     molecular oxygen as oxidizing agent, unless otherwise specified.              Preparations were assayed without further purification.                       .sup.2 The ELISA was conducted as per Table 1 with antisera raised to the     ST prepared as in (3) as the basis for comparisons. That ST preparation       was purified prior to coupling, while preparation (3) shows the results       for the unpurified preparation.                                               .sup.3 The suckling mouse assay was conducted as per Table 1 with             percentages being based upon the value for biologic ST, the values in         parentheses being nanograms of ST providing a gut: whole body ratio of        0.083.                                                                        .sup.4 N.D. = Not determined.                                                 .sup.5 It is believed that the antigenicity value obtained is anomolously     low.                                                                     

The above results illustrate that the highest antigenic activity (ELISA)is achieved under oxidation conditions in which the first syntheticpolypeptide is present in solution at a concentration of less than about2 milligrams per milliliter, oxygen in the air is the oxidizing agent,the pH value is below about 10, and particularly where the pH value isabout 7.5 to about 8.0, the temperature of the reaction is about zero toabout 25° C. and the oxidation reaction time is less than about 24hours.

PREPARATION OF MULTIMERIC ST A. Multimeric Synthetic ST Dimer (ST/ST)

A first polypeptide having the 36-residue sequence of two ST Ibmolecules joined head-to-tail by an amide bond was prepared by thestepwise, solid phase synthesis described before. The amino acid residuesequence of that material, taken from left to right and in the directionof amino-terminus to carboxy terminus, is shown in Formula IX, below:##STR14##

That molecule was then dissoled at a concentration of 1 milligram permilliliter in a 0.05 molar ammonium bicarbonate buffer having a pH valueof 8.0. The resulting solution was stirred gently in the presence ofatmospheric molecular oxygen as oxidant until free sulfhydryl groupscould not be detected with Ellman reagent, supra; i.e., overnight. TheST/ST molecule so prepared were separated from the remaining materialsin the reaction medium by column chromatography, freeze dried and werethen used without further purification. Amino acid analysis of the36-mer first polypeptide was consistent with the sequence shown inFormula IX.

The resulting ST/ST without further purification had an antigenicity toantibodies raised against natural, monomeric ST of 233 percent comparedto the antigenicity of the natural ST Ib to those same antibodies. Thebiological activity of this ST/ST was less than one percent of that ofnatural ST Ib in the suckling mouse assay, supra, up to the limits ofthe assay, here 640 nanograms.

Synthetic trimer (ST/ST/ST), tetramer (ST/ST/ST/ST), and longerhead-to-tail multimers of synthetic ST can be similarly prepared.Synthetic dimer and trimer ST molecules are particularly preferredbecause of the difficulties that attend in producing syntheticpolypeptides that contain more than a total of about sixty amino acidresidues.

Results using the synthetic dimer (ST/ST) in the preparation of avaccine are discussed in more detail hereinafter in Section V.

B. Multimeric Synthetic ST Polymer

The 18-mer first synthetic ST Ib polypeptide prepared as describedpreviously was used illustratively for the preparation of synthetic STpolymers whose plurality of synthetic ST repeating units are linkedtogether by intramolecular, interpolypeptide cystine disulfide bonds.

In an illustrative preparation, the polymeric synthetic ST (hereinaftersometimes referred to as P-ST) was prepared by dissolving the aboveprepared, first synthetic ST polypeptide (18-mer) in 0.1 molar ammoniumbicarbonate buffer having a pH value of 8.0 to provide a solution havinga concentration of about 1 milligram per milliliter of a first syntheticST polypeptide. Volumes of such solutions of 10 to 100 milliliters havebeen used. The solution so prepared was stirred gently at roomtemperature in an open beaker to provide oxidation of the Cysmercaptans. Analysis of the reaction medium after an oxidation period ofabout 16-24 hours using Ellman reagent, supra, indicated that no freesulfhydryl groups were present.

The oxidized reaction medium was lyophilyzed and the resulting, driedmaterial was resuspended in a 0.1 molar ammonium bicarbonate (pH8.0-8.5) buffer, and was placed upon a Sephadex G-50 chromatographycolumn equilibrated with same buffer for separation. A typicalseparation of polymeric synthetic ST (P-ST) from monomeric syntehtic ST(M-ST) is illustrated in the graph of FIG. 17 using such a column.

The ordinate of the graph is in units of optical density read at 278nanometers. The abscissa shows numbered fractions of 4.5 milliliterseach of eluate collected from the column. The eluate from fractionsnumbered 5-9 was collected as containing the P-ST, while fractionsnumbered 10-17 were collected as containing synthetic monomeric ST(M-ST).

Fractionation of P-ST so obtained using a column containing Bio Rad P-30(Bio Rad, San Raphael, CA) as the separating resin indicated that thevast majority of the P-ST isolated using the Sephadex G-50 column had anaverage molecular weight of at least about 40,000 daltons (40 kd P-ST).Further fractionation of the 40 kd P-ST using Bio Gel A-1.5 m resin (BioRad) indicated that most of that material had an average molecularweight of about 400,000 daltons, with some material having a molecularweight of greater than about 1,500,000 daltons (1500 kd P-ST). Stillfurther molecular weight fractionation using a column containing Bio GelA-5 m resin (Bio Rad) resolved the 1500 kd P-ST into one fraction thatincluded P-ST having a molecular weight of greater than about 15,000,000daltons (15,000 kd P-ST) and a second fraction having an averagemolecular weight of about 4,000,000 daltons (4000 kd P-ST).

The above molecular weights are approximations based upon a presumedglobular shape for the P-ST and a knowledge of the void volume andexclusion limits of the column, but without the use of internalstandards having known molecular weights. Each of the above-obtainedfractions was lyophilized after elution from the column.

The results desribed hereinafter (Sections V and VI) using P-ST are forlypophilized, Sephadex G-50 purified preparations that contain mostlypolymer whose average molecular weight is at least about 40,000 daltons;i.e., the 40 kd P-ST. Work is presently underway relating to theantigenicity, immunogenicity and biologic activity of fractions of P-SThaving varying molecular weights.

Typical preparations of 40 kd P-ST have an antigenicity to antibodiesagainst natural ST Ib of between 900 and 1500 percent of theantigenicity of the natural material (ST Ib), and have a biologicalactivity in the suckling mouse assay, supra, of about 20 percent or lessthan that of natural ST Ib.

GENERAL SYNTHETIC PROCEDURE

A general synthetic procedure for preparing a monomeric or multimericsynthetic ST molecule having at least about 10 percent of theantigenicity of biologic ST based upon the above results and severalother determinations is as follows:

(1) A monomeric, first synthetic polypeptide is prepared in thesubstantial absence of oxidizing agent. The first synthetic polypeptideincludes the amino acid residue sequences of Formula I, Formula III,Formula V, Formula VI or Formula IX, at least two Cys residues whoseR_(a-f) ¹⁻⁶ -groups are hydrogen; i.e., two CysH residues, and is freefrom any intramolecular, cystine disulfide bonds, and may contain anR_(m) ¹³ -group wherein "m" has a value of one.

(2) The first polypeptide so prepared is provided, and is dissolved ordispersed in an aqueous composition at a concentration of less thanabout 5 milligrams per milliliter, more preferably at a concentration ofless than about 2 milligrams per milliliter, and most preferably at aconcentration of about 1 milligram per milliliter to about 0.1milligrams per milliliter. The pH value of the composition into whichthe first polypeptide is dissolved or dispersed is preferably alkalineand less than about 10.5, and more preferably is about 7.5 to about10.5.

(3) The first polypeptide-containing composition is thereafter contactedwith molecular oxygen in the air as an oxidizing agent. The pH of thesolution during oxidation is preferably about 7.0 to about 9.5, and morepreferably about 7.5 to about 9, and most preferably about 7.5 to about8.0. The solution is preferably contacted with the oxidant by gentlestirring in a vessel open to the air.

(4) Contact between the composition and the air is maintained for aperiod of about 1 to about 24 hours, and more preferably for about 2 toabout 8 hours, to form at least one intramolecular, intrapolypeptide orinterpolypeptide cystine disulfide bond from the at least two Cys (CysH)residues present. For monomeric synthetic ST and head-to-tail STmultimers, the at least one cystine disulfide bond is an intramolecular,intrapolypeptide bond, while for the polymeric synthetic ST (P-ST) theat least one cystine disulfide is an intramolecular, interpolypeptidebond. It is preferred that each ST repeating unit of P-ST form anaverage of about two interpolypeptide cystine disulfide bonds so thatP-ST molecules having more than two ST repeating units are formed.

In preferred practice for monomeric synthetic ST, the disulfide bond isformed between the Cys residues preceding the pairs R_(g) ⁷ and R_(k)¹¹, R_(h) ⁸ and R_(j) ¹⁰, and R_(i) ⁹ and R_(l) ¹² of Formulas I, III, Vor VI, which correspond to the positions of the residues numbered 5 and10, 6 and 14, and 9 and 17 from the amino-terminus of the ST Ibmolecule, respectively. In more preferred practice, contact betweenmolecular oxygen and the solution is maintained for a period sufficientto form two disulfide bonds, preferably between the above-mentionedpairs of Cys residues, and still more preferably for a period sufficientto form three disulfide bonds, again preferably between said pairs ofCys residues.

The oxidation is preferably carried out at a temperature of about 0° C.to about 25° C.

(5) Upon completion of the oxidation reaction, the synthetic ST istypically collected as by lyophilization, and purified as by columnchromatography.

III. USES OF MONOMERIC SYNTHETIC ST

The monomeric synthetic ST prepared as discussed above was linked to acarrier molecule or used alone in ELISA measurements in studies carriedout under the direction of Dr. Frederick A. Klipstein of the Universityof Rochester Medical Center, Rochester, N.Y. The results of experimentaldeterminations and the procedures for carrying out these determinationsare discussed herein and in Section IV, hereinafter. Unless otherwisestated, the studies discussed in Sections III and IV were carried outwith the monomeric synthetic ST (M-ST) of this invention.

A. Properties

ST Ib purified to homogeneity from human E. coli LT³¹ /ST⁺ strain 18D(O42:H47) has an estimated molecular weight of 1,972 and consists of tendifferent amino acids arranged in a sequence of 18 amino acids [Stapleset al., supra] whose primary structure was shown by Chan et al., supra,to be AsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn. Asynthetic molecule with this primary structure was prepared (Section II)and the biological properties of this polypeptide were compared withthat of pure ST obtained by bacterial growth of strain 18 D (biologicST).

Capacity To Induce Fluid Secretion

Synthetic and biologic ST were substantially equally potent in theirabilities to induce fluid secretion in the suckling mouse assay (FIG. 1)and rat ligated ileal loops (FIG. 2). The slight variations observed inthe minimum effective dosages of the two toxins in each assay model werewithin the range of experimental variation, and, in fact, severalsubsequently prepared preparations of synthetic ST evoked an identicalresponse to that of the native or biologic ST in the suckling mouseassay. Exposure to 100° C. for 30 minutes did not affect the potency ofeither toxin in either animal assay system.

Previous observations have shown that destruction of the disulfidebridges of biologic ST by treatment with reducing reagents such as2-mercaptoethanol or dithiothreitol abolished its biologic activity,Staples, et al., supra. Exposure of synthetic ST to 5×10⁻⁴ molardithiothreitol for 60 minutes also abolished its secretory activity inthe suckling mouse assay.

Immunological Relationship

The antigenicities of synthetic and biologic ST were similar when eachwas tested by enzyme linked immunosorbent assay (ELISA) usinghyperimmune antisera to biologic ST. When those concentrations whichyielded an optical density of 0.600 at 410 nanometers were compared, theantigenicity of synthetic ST was observed to be 70% that of biologic ST(FIG. 3), but the antigenicity of the toxins was identical when bothwere tested using hyperimmune antisera to synthetic ST. These resultswere substantially improved in later preparations as illustrated inTable 1, above, with the synthetic ST being about 3-fold better thanbiologic ST Ib in this same ELISA assay.

In order to evaluate the neutralizing effect of hyperimmune antisera toeither synthetic or biologic ST on the secretory effect of the toxins inthe suckling mouse assay, three mice for each datum point were given 100microliters intraintestinally containing 2 mouse units (MU), twice theminimum effective dosage (see Materials and Methods Section IV A) ofeach toxin that had been incubated with the designated antiserumdilution for 3 hours at 37° C. (FIG. 4). The number of mouse unitsneutralized by 1 milliliter of anitserum was derived from multiplyingthe projected antiserum dilution required to neutralize (i.e., yield agut:carcass ratio of at least 0.083) the secretory effect times the10-fold dilution factor times the factor of 2 in order to adjust for the2 mouse units used.

Hyperimmune antisera to each of the toxin preparations seroneutralizedthe secretory effect in the suckling mouse assay of synthetic andbiologic ST to the same approximate degree: one milliliter ofhyperimmune rabbit antiserum to biologic ST neutralized 160 MU ofsynthetic and 190 MU of biologic ST, while one milliliter of goathyperimmune antiserum to synthetic ST neutralized 220 MU of syntheticand 240 MU of biologic ST.

Immunization of Rats

Immunization with the synthetic ST yielded serum antitoxin titers of1:32 (four-fold greater than that of the controls) and mucosal IgAtiters of 1:64 (five-fold greater than that of the controls). Fluidsecretion was reduced by a significant degree that was comparable to anamount previously observed in rats immunized with semipure biologic ST[Klipstein, et al., Infect. Immun., 34: 637-639 (1981)] in ratschallenged with either synthetic or biologic ST and with the viableST-producing strain (Table 3), below.

                  TABLE 3                                                         ______________________________________                                        Results Of Challenge In Immunized Rats                                                   Challenge .sup.a                                                   ST immunogen ST(B) toxin                                                                             ST(S) toxin LT.sup.- /ST.sup.+                         ______________________________________                                        Synthetic(S) 54 ± 2 66 ± 2   55 ± 1                                  Biologic (B).sup.b                                                                         83 ± 9 ND.sup.c    69 ± 2                                  ______________________________________                                         .sup.a Mean ± standard error of the mean percent reduced secretion in      immunized rats as compared to similarly challenged unimmunized animals.       .sup.b Data taken from Klipstein et al., Infect. Immun., 34:637-639           (1981).                                                                       .sup.c Not determined.                                                   

The above results show that immunization with a synthetically-producedST toxin whose structure is based on that of human ST providesprotection against challenge with ST-producing enterotoxigenic strainsof E. Coli of human origin.

Further details of the studies discussed in this section (III A) may befound in Klipstein et al., Infect. Immun., 39: 117-121 (1983), whosedisclosures are incorporated herein by reference.

B. ST-LT Conjugates

The results discussed in this section relate to conjugate compositionsand their properties obtained by cross-linking, under variousconditions, synthetic ST (Section II) to either the LT holotoxin or itsnontoxic component, the B subunit as a carrier which is responsible forbinding of the toxin to specific receptors on the mucosal surface. Theefficacy of a vaccine consisting of synthetic ST cross-linked to the Bsubunit for arousing specific serum and mucosal antitoxin responses toeach of the component toxins is also demonstrated, thus providing strongprotection in immunized rats against challenge with either toxin form orheterologous viable ETEC which produce either ST or LT.

Conjugation of ST to LT

Synthetic ST was conjugated to the LT holotoxin from an initial molarratio of ST to LT of 100:1 using ratios of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) to total conjugateprotein which varied between 0.5:1 to 40:1 (FIG. 5). Coupling of themaximum amount of ST to LT occurred at an EDAC to conjugate ratio of1:1; however, an unacceptable degree of residual LT toxicity persistedat this ratio. Increasing the EDAC to conjugate protein ratio resultedin a progressive decrease in residual LT toxicity but this wasaccompanied by a corresponding reduction in the antigenicity of bothtoxins in the conjugate.

An EDAC to conjugate ratio of 20:1 resulted in a conjugate with anacceptable degree of reduced LT toxicity (0.3%), but the antigenicity ofboth component toxins was reduced to less than 25% at this ratio,indicating that this conjugate would be an ineffectual antigen. Theseobservations led to substitution of the nontoxic B subunit for LT,thereby circumventing the problem of residual LT toxicity.

Conjugation of ST to B Subunit

Synthetic ST was conjugated to B subunit from an initial molar ratio ofST to B subunit of 100:1, using EDAC to conjugate protein ratios whichvaried between 0.5:1 and 40:1. The pattern of ST incorporation and thechanges in antigenicity were similar to those noted for ST conjugationto LT except that the maximum amount of ST was coupled at an EDAC toconjugate ratio of 2:1. The toxicity of the conjugate obtained at thisratio was confined to that of the ST present; it was reduced to 0.3% ofunattenuated toxin.

An EDAC to conjugate ratio of 2:1 was employed, therefore, in studieswhich utilized radiolabelled ST to determine the influence of theinitial molar ratio of ST to B subunit on the amount of ST incorporatedinto the conjugate. Ten nanomoles of ST (5 nanomoles of cold, unlabelledST plus 5 nanomoles of radiolabelled ST) were mixed with from 2 to 0.1nanomoles of B subunit yielding initial ST to B subunit molar ratios offrom 5:1 to 100:1 (FIG. 6). Increasing the initial ST to B subunit ratioresulted in progressively greater amounts of ST coupled to the Bsubunit. Although an initial ST:B subunit ratio of 25:1 yielded a finalconjugate which contained more than 90% ST on a molar basis, theproportion of ST was only 25% on a weight basis; the latter valueincreased when larger initial molar ratios were used.

Antigenicity of the Conjugates

The effect of the EDAC concentration on the composition and antigenicityof the individual toxins in the final conjugates was assessed byconjugating a 50:1 molar ratio of ST to B subunit using EDAC toconjugate ratios which varied between 1:1 and 2:1 (FIG. 7). Maximumconjugation of ST occurred at an EDAC to conjugate ratio of 2:1; howeverratios of greater than 1.5:1 resulted in a precipitous fall in STantigenicity and a moderate decline in B subunit antigenicity. Themaximum number of ST antigen units (derived by multiplying thepercentage of toxin present times the percentage of its antigenicity)was achieved at an EDAC to conjugate ratio of 1.5:1.

An EDAC to conjugate ratio of 1.5:1 was used, therefore, in studieswhich determined the effect of the initial molar ratio of ST to Bsubunit on the composition and properties of the final conjugate.Increasing the initial molar ratio of ST to B subunit from 50:1 to 100:1resulted in a progressively greater proportion of ST in the finalconjugate (FIG. 8).

Since changes in the initial molar ratio were achieved by keeping theamount of ST (300 nanomoles) constant and reducing the amount of Bsubunit added (from 6 to 3 nanomoles), the ratio of EDAC to ST decreasedwhile the ratio of EDAC to B subunit rose as initial molar ratiosincreased. This change accounted for the fact that the ST antigenicityrose slightly while that of the B subunit fell moderately. The neteffect of these two factors (composition and antigenicity) was thatincreasing the initial molar ratio resulted in progressively greateramounts of ST antigen units, with a corresponding fall in B subunitantigen units, in the final conjugate.

Properties of The Vaccine Used For Immunization

It was previously shown that the B subunit is a weaker antigen than theLT holotoxin on a molar basis [Klipstein et al., Infect. Immun., 31:144-150 (1981)] and preliminary studies indicate that it is a weakerantigen in terms of antigen units than either the LT holotoxin orsynthetic ST. This led to selection of a vaccine which contains more Bsubunit than ST antigenicity.

The antigen was produced by conjugating an initial ST to B subunit molarratio of 50:1 using an EDAC to conjugate ratio of 1.5:1. The conjugatecontained 36% (by weight) ST which had 85% retained antigenicity and0.13% persistent toxicity, and 64% (by weight) B subunit which retained89% of its antigenicity. When tested directly (i.e., in samples notadjusted to contain 100% of each toxin), the vaccine contained 37 ST and59 B subunit antigen units per 100 micrograms and had a residual STtoxicity of 0.06%.

Results of immunization

Rats immunized with the above vaccine were given 1,000 microgramsprimary immunization by the intraparenteral (i.p.) route followed by thetwo 3,000 microgram peroral (p.o.) boosts. Previous studies have shownthat in rats immunized with LT by this approach, the degree of theantitoxin response and of protection correlate with the total p.o.dosage [Klipstein et al., Infect. Immunol., 31: 144-150 (1981);Klipstein et al., Infect. Immunol., 37: 1086-1092 (1982); Klipstein etal., Infect. Immunol., 31: 252-260 (1981)]. This immunization scheduleamounted to 2,200 ST and 3,450 B subunit antigen units.

Serum IgG antitoxin titers to ST were increased 4-fold and those to theB subunit were increased 5-fold over values in control, unimmunizedrats. Mucosal secretory IgA antitoxin titers to both ST and B subunitwere 7-fold greater in immunized rats than in controls. Immunized ratswere significantly protected against challenge with LT or with eithersynthetic or biologic ST as well as against heterologous viableorganisms which produce these toxins either singly or together as shownin Table 4, below.

                  TABLE 4                                                         ______________________________________                                        Results of Challenge in Rats Immunized With                                   Cross-Linked ST-B Subunit Vaccine                                             Percent Reduced Secretion After Challenge With:.sup.a                                                    ST(B) ST(S)                                        LT toxin                                                                             LT.sup.+ /ST.sup.-                                                                      LT.sup.+ /ST.sup.+                                                                      toxin.sup.b                                                                         toxin.sup.b                                                                         LT.sup.- /ST.sup.+                     ______________________________________                                        94 ± 3                                                                            61 ± 2 68 ± 2 97 ± 3                                                                           78 ± 1                                                                           76 ± 2                              0.5 ng.sup.c                                                                         5 ng.sup.c                                                                              5 ng.sup.c                                                                              10.sup.8,d                                                                          10.sup.8,d                                                                          10.sup.8,d                             ______________________________________                                         .sup.a Values are the mean ± standard error of the mean. Reduced           secretion of more than 50% represents a significant (P less than 0.001)       difference between immunized and unimmunized rats                             .sup.b ST(B) signifies biologic ST, and ST(S) signifies synthetic ST.         .sup.c Amount of challenging toxin in nanograms (ng).                         .sup.d Number of challenging organisms.                                  

The above findings that synthetically produced, purified ST can be usedto provide an effective, nontoxic antigen when it is cross-linked to theLT toxin B subunit surmounts a major obstacle in the development of asafe, practical vaccine that provides protection against ETEC strainswhich produce either the ST or LT form of toxin. Previous results showedthat cross-linking a semipure preparation of biologic ST to LT yieldedan antigen in which the ST acquired antigenicity as a function ofcoupling to the large molecular weight LT molecule, and in which, underthe proper conjugation conditions, most of the antigenicity of thecomponent toxins was maintained while their toxic properties weregreatly reduced [Klipstein et al., Infect. Immunol., 37: 550-557(1981)].

Although that vaccine with biologic ST provided strong protection inimmunized animals against challenge with ETEC strains which produceeither toxin form, the heterogeneous composition of the semipure STtoxin component clearly precluded its adoption for human use. Thecomplicated and tedious methodology involved in processing biologic STto total purity renders large scale, much less commercial scale,production of this material difficult. The above findings indicate thatsynthetically-produced ST, which can readily be made in largequantities, provides an equally effective vaccine.

The reaction conditions for conjugates derived from synthetic ST whichyield maximal incorporation of ST with the carrier together with optimalproperties in terms of residual antigenicity and toxicity differ fromthose previously observed for conjugation of semipure biologic ST to LT.In both circumstances, (1) a critical amount of the conjugating reagentcarbodiimide was necessary for coupling the maximum amount of ST to thecarrier, (2) the proportion of ST present in the final conjugate wasdependent on the initial molar ratio of ST mixed with LT, and (3)increasing the ratio of carbodiimide to either toxin in the conjugateresulted in a progressive decline both in the antigenicity and intoxicity of the cross-linked toxins. In the case of semipure biologicST, conjugation conditions were identified which yielded a conjugatewith maximal incorporation of ST to LT and at the same time retainedmost of the antigenicity but markedly reduced the toxicity of both ofthe cross-linked toxins.

Such did not occur, however, when synthetic ST was conjugated to LT.Maximum coupling of synthetic ST to LT occurred at a much lowercarbodiimide to conjugate ratio under which conditions the residual LTtoxicity of this conjugate was unacceptably high. A reduction in LTtoxicity to acceptable levels was achieved only at carbodiimide to toxinratios which severely compromised the antigenicity of both of thecross-linked toxins. These findings led to circumventing this problem bysubstituting the nontoxic B subunit for the LT holotoxin as the carrier.

The proportion of antigenicity (expressed as antigen units) for each ofthe component toxins present in the final conjugate derived bycross-linking synthetic ST to the B subunit can be altered by varyingthe conditions of the conjugation reactions. Thus, in the presence ofthe proper concentration of carbodiimide, a low initial molar ratio ofST to B subunit yielded a conjugate with predominantly ST antigenicitywhereas a high initial molar ratio yielded one in which B subunitantigenicity is greatest.

Preliminary observations suggested that synthetic ST is a more effectiveantigen than the B subunit. This led to selection of a cross-linkedantigen for evaluation by immunization in rats that contained roughlyone-third ST and two-thirds B subunit antigenic activity. When given inlarge p.o. doses, that vaccine aroused at least a 4-fold serum andmucosal antitoxin response against both component toxins, thus providingsignificant protection against challenge by either the ST or LT toxinsand heterologous viable bacteria which produce either toxin form.

Since all ETEC strains evoke diarrhea through the elaboration of the LTor ST toxins, either singly or together, the arousal of a sufficientlystrong antitoxin response to each of these toxins provides uniformlyeffective protection against all ETEC strains irrespective of thesomatic serotype, specific fimbrial antigen or type of toxin produced.Such was shown to be the case in the above study among rats immunizedwith the cross-linked ST-B subunit vaccine.

Immunization with LT given exclusively by the parenteral route arousedonly a serum IgG antitoxin response which provided only transientprotection in rats, whereas p.o. booster immunization yielded extendedprotection due to the arousal of mucosal secretory Ig antitoxin.[Klipstein et al., Infect. Immun., 37: 1086-1092 (1982) and Klipstein etal., Infect. Immun., 27: 81-86 (1982)].

Mucosal secretary IgA antitoxin titers to both ST and the B subunit inrats immunized perorally with the cross-linked vaccine in the abovestudy exceeded those previously found necessary to provide extendedprotection in rats immunized with just LT. This makes it clear that thecross-linked vaccine should be given by the p.o. route. The above dataare insufficient to determine whether primary immunization by theparenteral route is a prerequisite for subsequent effective p.o.immunization since such has been found to be the case for rats immuzinedwith LT [Klipstein et al., Infect. Immun., 31: 144-150 (1981); Klipsteinet al., Infect. Immun., 37: 1086-1092 (1982); and Klipstein et al.,Infect. Immun., 27: 81-86 (1980)] and rats and dogs immunized withcholera toxoid [Pierce et al., Infect. Immun., 21: 185-193 (1978);Pierce et al. J. Infect. Dis., 135: 888-896 (1977)].

Further details of the studies discussed in this section (III B) may befound in Klipstein et al., J. Infect. Dis., 147: 318-326 (1983), whosedisclosures are incorporated herein by reference.

C. Synthetic ST Immunizations in Rats Immunogenicity of ST and the Bsubunit

Rats were immunized with graded antigen unit dosages of either the Bsubunit or synthetic ST coupled to porcine immunoglobulin G (PIG;Materials and Methods section C) as is shown in FIG. 9. Those rats givenvariable dosages of the i.p. primary immunization all received two p.o.booster immunizations of 1000 antigen units each, and those givenvariable dosages for the p.o. boosters all received i.p. primaryimmunization with 200 antigen units. Rats immunized with ST werechallenged with human LT⁻ /ST⁺ strain Tx 452 and those immunized withthe B subunit were challenged with human LT⁺ /ST⁻ strain PB 258.

Increasing the antigen unit dosages of either the i.p. primryimmuniztion or the p.o. boosters of either antigen resulted in parallelincreases in antitoxin titers and in the degree of protection againstchallenge with the respective organisms. Values for serum IgG antitoxintiters rose proportionately but were consistently one or two-fold lessthan mucosal IgA antitoxin titers.

Dosages of either antigen of 100 antigen units for i.p. primaryimmunization and a total of 2000 antigen units for the p.o. boosters(i.e., 2 boosters of 1000 antigen units each) were required to achieveat least 4-fold increases in mucosal IgA antitoxin titers andsignificant protection (i.e., greater than 50% reduced secretion)against challenge with the respective LT- pr ST-producing strain.

Protection against an LT⁺ /ST⁺ strain

In order to determine the minimum antigen unit dosage of ST or B subunitnecessary to achieve strong protection against a strain which producesboth LT and ST, rats were immunized with each toxin separately, using ani.p. primary immunization of 200 antigen units followed by variableantigen unit dosages of the p.o. boosters, and were challenged both withthe respective LT- or ST-producing strains and with human LT⁺ /ST⁺strain H 10407 (FIG. 10).

Strong protection against the LT⁺ /ST⁺ strain was achieved only by atotal p.o. booster dosage of 2000 antigen units. In each instance, thedegree of protection against the LT⁺ /ST⁺ strain was less than that forthe strain which produced only the single homologous toxin.

Conjugation conditions for the vaccine

The preceding observations indicate that the optimal vaccine shouldcontain equal antigenic proportions of each component toxin. It wasshown in Section B, hereinabove, that when the ratio of carbodiimide tototal conjugate protein is kept constant at 1.5:1 by weight, increasingthe initial molar ratio of ST mixed with the B subunit yields a finalconjugate with progressively more ST and less B subunit antigenicity.

When the initial molar ratio of ST to B subunit was varied between about60:1 to about 75:1, it was found that a ratio of about 70:1 resulted ina conjugate which consisted of 50% of each toxin component by weight andcontained 460 ST and 440 B subunit antigen units per milligram (FIG.11). All subsequent conjugates discussed in this section were preparedin this manner. In five consecutive lots, mean antigen units permilligram were 474 for ST and 460 for the B subunit using the aboveratios.

Properties of the vaccine

(i) Immunogenicity.

In order to confirm the fact that the immunogenicity of the toxincomponents cross-linked in vaccine form is the same as that of theindividual components, rats were immunized by i.p. primary immunizationof 200 ST antigen units followed by graded p.o. booster dosages of STgiven either in vaccine form or coupled to the immunologicallynonspecific carrier PIG. The results are shown in FIG. 12. As seen inFIG. 12, the antitoxin response and degree of protection against thehuman LT⁻ /ST⁺ strain were substantially identical in rats immunizedwith either form of ST conjugate.

(ii) Toxicity.

Assay of graded amounts (by protein content) of ST alone or of thevaccine in suckling mice showed that the vaccine contained 0.14 mouseunits of ST activity per microgram, which represented 0.08% of the valueof 175 mouse units per microgram of ST alone (FIG. 13). When gradedamounts of either ST alone or the vaccine were tested in rat ligatedileal loops, the ED₅₀ of the vaccine, 2.6 micrograms, was 0.08% that ofthe value of the 2.0 nanograms for ST alone (FIG. 14). The B subunitalone had an ED₅₀ of 95 nanograms in ligated ileal loops which was 0.2%of the value of 0.19 nanograms for the LT holotoxin. Whether thissecretory activity was due to the B subunit itself or a manifestation ofslight, otherwise undetected contamination with LT is uncertain.

In order to determine whether the B subunit component was contributingto the residual secretory activity of the vaccine in the rat ligatedileal loop assay, graded dosages of vaccine were tested after heatinactivation of the B subunit (or its LT contaminant) by exposure to 65°C. for 1 hour. The secretory response to heated and unheated vaccine wasthe same, excluding a role for the B subunit in this response. Theseobservations indicate that the toxicity of a dosage of vaccinecontaining 1000 antigen units of each component toxin would consist ofthe equivalent of 1.7 micrograms of unattenuated ST.

(iii) Protection against human and porcine strains.

Rats were immunized by primary i.p. immunization with vaccine containing200 antigen units of each component toxin and two p.o. boosts, each ofwhich had 1000 antigen units of each toxin component. This raised serumIgG antitoxin titers to both toxin components by 3-fold and mucosal IgAantitoxin titers by 5-fold to ST and by 6-fold to the B subunit. Theimmunized rats were significantly protected (P less than 0.001) againstchallenge with viable human or porcine strains which produce LT or STtoxin, either singly or together, as is shown in Table 5, below.

                  TABLE 5                                                         ______________________________________                                        Results of Challenge In Rats Immunized                                        With the Cross-Linked Vaccine                                                                                      % Reduced                                Source  Toxicity   Strain    Serotype                                                                              Secretion                                ______________________________________                                        Human   LT.sup.+ /ST.sup.-                                                                       PB 257    015:H.sup.-                                                                           74 ± 1                                Porcine LT.sup.+ /ST.sup.-                                                                       P 263     08:H19  72 ± 3                                Human   LT.sup.+ /ST.sup.+                                                                       H 10407   078:H11 61 ± 1                                Porcine LT.sup.+ /ST.sup.+                                                                       P 1362    0149:H?.sup.b                                                                         73 ± 2                                Human   LT.sup.- /ST.sup.+                                                                       Tx 452    078:H12 79 ± 2                                Porcine LT.sup.- /ST.sup.+                                                                       P 987     09:H.sup.-                                                                            81 ± 2                                ______________________________________                                         .sup.a Mean ± standard error of the mean percent reduced secretion in      immunized rats as compared to similarly challenged unimmunized animals.       Values of more than 50% represent a significant (P less than 0.001)           difference between the two groups.                                            .sup.b The complete identification of this serotype is uncertain.        

Primary parenteral immunization was given to an additional group of ratsby the subcutaneous (s.c.) route using alum as the adjuvant, prepared asdescribed previously for LT immunization [Klipstein et al., Infect.Immun., 37: 1086-1092 (1982)]. Since this approach has been found torequire twice the dosage used for the i.p. route for effective LTprimary immunization, the s.c. dosage of the vaccine given was doubledto 400 antigen units; the p.o. dosage was unchanged at 1000 antigenunits. This raised at least 5-fold mucosal IgA antitoxin titers to bothtoxin components and provided significant protection against challenge,with secretion reduced by 77±3% against the human LT⁺ /ST⁻ strain and by71±2% against the human LT⁻ /ST⁺ strain.

The above results indicate that, when evaluated in the manner ofKlipstein et al., Infect. Immun., 31: 144-150 (1981), the antigenicityof synthetically-produced ST is substantially the same as that of the Bsubunit. Essential to this comparison was the expression of dosage ofconjugated toxin in terms of antigen units rather than on a weightbasis.

This information led to modifying the conjugation conditions usedpreviously to cross-link synthetic ST to the B subunit, Section B above,in order to produce a vaccine that contains equal antigenic proportionof ST and B subunit. The antigenicity of the synthetic ST component invaccine form was shown to be identical to that of this toxin when givencoupled to a nonspecific immunoglobulin carrier. Immunization of ratswith vaccine, given at those antigen unit dosages found effective foreach of the component toxins given seprately, raised a strong antitoxinresponse to each of the component toxins and provided significantprotection against viable ETEC strains that produce LT or ST, eithersingly or together. Observations derived from immunizations with eachtoxin component given separately indicated that the protection afforedby the vacine against the LT⁺ /ST⁺ strain was attributable to both toxincomponents.

Frantz and Robertson have reported that antisera to porcine ST reactswith ST from ETEC strains of porcine, bovine, and human origin [Infect.Immun., 33: 193-198 (1981)]. The above results indicate thatcross-protection can be achieved by immunization with either toxinirrespective of its source. Thus, immunization with the cross-linkedvaccine containing a B subunit derived from porcine LT and synthetic STbased on the structure of human ST provided equally strong protectionagainst human and porcine LT- and ST-producing strains.

Immunization was given in the above study by means of parenteral primaryimmunization followed by p.o. boosters because it was previously foundin the rat animal model that, (1) parenteral priming is a prerequisitefor strongly effective p.o. booster immunization, (2) only p.o.immunization raises mucosal IgA antitoxin titers, and (3) extendedprotection is achieved only when a sufficient p.o. dosage is given thatraises mucosal IgA titers by at least 4-fold. Immunization with thecross-linked vaccine by this approach yielded increases of thismagnitude in mucosal IgA antitoxin titers to both of the componenttoxins. The subcutaneous (s.c.) route and alum adjuvant were shown to beequally effective for immunization.

Further details of the studies discussed in this section (III C) may befound in Klipstein et al., Infect. Immun., 40: 924-929 (1983), whosedisclosures are incorporated herein by reference.

D. Synthetic ST Immunization in Rats and Rabbits

The above discussed results (Section III C) were obtained in the ratusing i.p. immunizations followed by p.o. boosters. The resultsdiscussed below were obtained in both rats and rabbits using the peroralroute of administration for both the primary and booster immunizations.

The results below indicate that the peroral route of immunization isequally as effective as is the i.p. route. In addition, it is noted thatthe vaccine did not cause diarrhea in any animal when given by the p.o.route, nor did it cause fluid secretion when instilled into rabbitligated ileal loops.

Rat Studies

(i) i.p./p.o. immunization.

Rats received primary immunization with 200 AU (antigen units; seeMaterials and Methods Section IV D) of vaccine given i.p. with Freund'scomplete adjuvant (FCA) followed by two p.o. boosters of 1000 AU each.This dosage was selected because it has been shown (Section C, supra) tobe the minimal amount necessary to provide significant (P less than0.001) protection against challenge with viable strains which produceeither toxin form. This immunization raised 4-fold increases in serum,and at least 6-fold increases in mucosal, antitoxin titers to each toxincomponent of the vaccine (Table 6, below) and it provided protectionindex (PI) values of 3.4 against challenge with LT and 4.0 againstchallenge with ST (FIG. 15).

                  TABLE 6                                                         ______________________________________                                        Antitoxin Response And Degree Of                                              Protection In Immunized Rats                                                         Antitoxin  Antitoxin                                                          to B.sup.a to SI        PI                                             Route of                               vs   vs                                Immunization                                                                           Serum   Mucosal  Serum Mucosal                                                                              LT   ST                                ______________________________________                                        i.p./p.o.                                                                              4       6        4     7      3.4  4.0                               ______________________________________                                         .sup.a Values are the fold increase in the reciprocal of the geometric        mean titer in immunized over control animals.                                 .sup.b PI = protection index.                                            

(ii) Other Immunization Approaches

In order to determine the effectiveness of other parenteral routes,adjuvants and p.o. delivery systems, additional groups of four rats eachwere given primary immunization with 400 AU of vaccine by thesubcutaneous (s.c.) route using alum as the adjuvant, prepared asdescribed previously for LT [Klipstein et al., Infect. Immun., 37:1086-1092 (1982)]; this was followed by two p.o. boosters, each of 1000AU, given either 2 hours after p.o. cimetidine or in the form ofpH-dependent microspheres without pretreatment with cimetidine. Whenchallenged with LT⁻ /ST⁺ strain Tx 452, each of these alternativeapproaches to immunization yielded the same significant (P less than0.001) degree of reduced secretion as that achieved by using the i.p.route with FCA followed by p.o. boosters given after cimetidine. Theseresults are shown in Table 7, below.

                  TABLE 7                                                         ______________________________________                                        Effectiveness of Alternative                                                  Routes, Adjuvants And Delivery                                                Systems Of The Vaccine in Rats                                                Primary       Booster      Protection                                         Route/Adjuvant                                                                              Route/Protection                                                                           vs LT.sup.- /ST.sup.+a                             ______________________________________                                        i.p./FCA      p.o./cimetidine                                                                            79 ± 2                                          s.c./alum     p.o./cimetidine                                                                            71 ± 2                                          s.c./alum     p.o./microspheres                                                                          67 ± 2                                          ______________________________________                                         .sup.a Mean ± SEM percent reduced secretion in immunized animals as        compared to similarly challenged unimmunized controls. Values of more tha     50% represent a significant (P less than 0.001) difference between the tw     groups.                                                                  

Rabbit Studies

(i) i.m./p.o. immunization

Four rabbits received primary immunization with 500 AU of vaccine givenintramuscularly (i.m.) with FCA, followed by two p.o. boosters of 1000AU each. This raised more than 5-fold increases in serum, and 4-foldincreases in mucosal antitoxin titers to each component of the vaccine.PI values were more than 9 against challenge with either LT or ST. Thesedata are shown in FIG. 16, and in Table 8, below.

                  TABLE 8                                                         ______________________________________                                        Antitoxin Response And Degree Of                                              Protection In Immunized Rabbits                                                      Antitoxin  Antitoxin                                                          to B.sup.a to ST        PI                                             Route of                               vs   vs                                Immunization                                                                           Serum   Mucosal  Serum Mucosal                                                                              LT   ST                                ______________________________________                                        i.m..sup.c /p.o.                                                                       5       4        6     4      9.3  10.0                              p.o./p.o.                                                                              3       5        2     4      8.6   8.1                              ______________________________________                                         .sup.a Values are the fold increase in the reciprocal of the geometric        mean titer in immunized over control animals.                                 .sup.b PI = protection index.                                                 .sup.c i.m. = intramuscular.                                             

(ii) p.o./p.o. immunization

Four rabbits received immunization with 1000 AU of vaccine given p.o. onthree occasions. This raised 4-fold increases in mucosal, but not inserum, antitoxin titers and provided strong protection, with PI valuesof more than 8 against challenge with either toxin form. These data arealso shown in Table 8, above.

Toxicity of the Vaccine

Previous studies (Section III C) have shown that the toxicity of the STcomponent of the vaccine is reduced to 0.15% of unattenuated toxin. Adosage of 1000 AU of vaccine would thus contain the equivalent of 1.7micrograms of unattenuated ST (0.15% times the 50% ST component of 2.2milligrams). The toxicity of this dosage of the vaccine and of largeramounts of unattenuated ST was evaluated in unimmunized animals. (i) Thep.o. administration of 1000 AU of vaccine to eight rabbits and 20 ratsproduced no adverse effects such as diarrhea, and the instillation ofthis dosage of vaccine into four ligated ileal loops in two rabbitsfailed to evoke any fluid response. (ii) The p.o. administration of 250micrograms of unattenuated ST to two rabbits and rats did not causediarrhea. The instillation of 25 micrograms of unattenuated ST did notcause any fluid secretion in ligated ileal loops of four rabbits; adosage of 50 micrograms was required to yield a positive fluid:lengthratio of 1.1±0.3 (mean±SEM).

The results of the above study establish the effectiveness ofimmunization with a vaccine made using a synthetic ST of this inventionin an experimental animal model, rabbits, in addition to rats.Protection in both animal models was demonstrated by use of the ligatedileal loop technique. The applicability of this technique to protectionunder conditions in which the entire intact intestine is acutelycolonized by enterotoxigenic strains of E. coli has been confirmed inrats immunized with LT [Klipstein et al., Infect. Immun., 28: 163-170(1980)].

The same p.o. dosage of vaccine (1000 AU), given after primaryparenteral immunization, resulted in a considerably stronger degree ofprotection, as manifested by PI values, in rabbits than in rats. Thisdifference may in part be attributable to the longer interval betweenimmunizations used for rabbits (14 days versus four days in rats), andperhaps to differences in sensitivity to toxin challenge. It alsoprobably indicates that rabbits are more responsive to immunization withthe vaccine than rats. This dosage used was the minimum found requiredto achieve significant protection against viable enterotoxigenic strainsin rats (Section C, supra).

The fact that this dosage was also effective in providing strongprotection in a larger experimental animal points to utility in animalhusbandry and humans. This is also suggested by the observations ofSvennerholm et al. who found that a p.o. immunization dosage of 500micrograms of cholera toxin B subunit is sufficient to arouse asignificant intestinal IgA antitoxin response in human volunteers[Lancet, 1: 305-308 (1982)].

The results of the present study indicate that exclusive p.o.immunization of rabbits with the synthetic ST-B vaccine achieved thesame strong degree of protection as that achieved by p.o. boosterimmunizations following parenteral primary immunization.

Further details of the studies discussed in this section (III D) may befound in Klipstein et al., Infect. Immun., 40: 888-893 (1983), whosedisclosures are incorporated herein by reference.

E. Cross Reactivity With Klebsiella ST Enterotoxin

Diarrheal epidemics among nursery children have implicated Klebsiellapneumoniae as the causing agent. The enterotoxigenicty of severalKlebsiella strains has been established by assay of cell-free culturefiltrates in rabbit ileal loops, in Yl adrenal cell or Chinese hampsterovary tissue culture assays for the heat-labile toxin (LT) and in thesuckling mouse assay, supra, for the heat-stable toxin (ST). TheKlebsiella LT and ST enterotoxins identified in these assays have notbeen purified, and their relationship to similar toxins produced E. coliis unknown.

Klipstein et al., J. Infect. Dis., 42: 838-841 (1983) report upon thepurification of Klebsiella ST toxin to apparent homogeneity, and uponits immunological relationship to E. coli ST. The disclosures of thatpublication are incorporated herein by reference.

Natural enterotoxins were obtained from Klebsiella strains TS 9(serotype 19), were isolated from the small bowel of a Puerto Ricanpatient having tropical sprue whose culture filtrate had previously beenshown to evoke a positive response in a suckling mouse assay, and werealso obtained from the E. coli strain 18 D (042:H47), an ST-onlyproducing strain isolated from the stool of a child with acute diarhea.The toxins were purified by the methods described by Staples et al., J.Biol. Chem., 255: 4716-4721 (1980) for the purification of human ST fromE. coli strain 18 D.

After three consecutive chromagraphic separation procedures used forpurification, the resulting toxin had been purified by a factor of 148over the originally obtained material, and thin layer chromotagraphy ofthe purified material showed a single band. The purified Klebsiella STwas equally potent as E. coli ST in the suckling mouse assay, within thelimits of experimental variation for that assay. In addition, treatmentof the Klebsiella ST with 5×10⁻⁴ molar dithiothreitol for 60 minutesabolished its secretory activity in the suckling mouse assay, as occurswith E. coli ST. Using a double sandwich ELISA as discussed in SectionIII A above, IV A hereinafter and in Klipstein et al., Infect. Immun.39: 117-121 (1983), the antigenicity of the Klebsiella ST was found tobe 69 percent that of E. coli ST.

Because of the functional similarities between E. coli ST and KlebsiellaST, it was of interest to determine whether a vaccine effective againstE. coli ST would also offer protection against Klebsiella ST. Thevaccine containing a conjugate of the E. coli LT B subunit and syntheticST that is described in Section III D hereinabove was utilized for theseimmunizations.

Sprague-Dawley rats weighing between 150 and 175 grams were givenprimary immunization with the vaccine containing 200 antigen units ofboth ST and LT B subunit by the intraperitoneal route with completeFreund's adjuvant. This was followed at four day intervals by two peroral booster immunizations of 1000 antigen units each, given two hoursafter the per oral administration of cimedeine (TAGAMET® available fromSmith Kline and French Laboratories, Carolina, Puerto Rica) at a dosageof 50 milligrams per kilogram of body weight in order ablate gastricsecretion.

Unimmunized control rats and immunized rats four to six days after thefinal booster immunization were challenged by the instillation of gradeddoses of the ST toxin in ligated ileal loops for 18 hours as describedin Section III D hereinabove, Section IV D and in Klipstein et al.,Infect. Immun., 49: 888-893 (1983). Each datum point for fluid secretion(presented as the means ± standard error of the mean) was derived fromchallenge in four or five immunized and five control rats. Theprotection index (PI) was determined by dividing that dosage of toxin inimmunized animals which yielded same secretion as the 50% effective dose(ED₅₀) in unimmunized animals by the value for unimmunized animals.

The results of this determination are shown graphically in FIG. 18wherein the data for the challenge with E. coli ST in immunized rats isthat shown in the lower portion of the graphs of FIG. 15, while the datarelating to challenge with Klebsiella ST are those generated in thisstudy. As can be seen the protection index (PI) against challenge withE. coli ST was 4.0 while that against Klebsiella ST was 2.6. The resultsshown in the graph of FIG. 18 illustrate that a vaccine containing aconjugated synthetic ST of this invention also offers some protectionagainst the ST enterotoxin produced by Klebsiella pneumoniae.

IV. MATERIALS AND METHODS FOR MONOMERIC ST Section A

Enterotoxin Production

The complete procedure for synthesis and purification of the syntheticST used herein and in each following lettered section is described indetail in Section II.

Biologic ST was purified to homogeneity from culture filtrates of strain18D by a modification [Klipstein et al. Infect. Immun., 37: 550-557(1982)] of the methods described by Staples et al., supra. The amountsof toxins were based on their protein concentration determined by themethod of Lowry et al., J. Biol. Chem., 193: 265-275 (1951).

Assay of Secretory Potency

The ability of graded dosages of the toxins to cause secretion wastested in the suckling mouse and rat ligated ileal loop assays usingpublished methods [Giannella, Infect. Immun., 14: 95-99 (1976) andKlipstein et al., Infect. Immun., 34: 637-639 (1981)]. One mouse unit(MU) in the suckling mouse assay is defined as that amount of toxinwhich yields an intestinal (gut):carcass weight ratio of at least 0.083.

Production of Hyperimmune Antiserum

Hyperimmune antiserum was raised in goats and rabbits to biologic ST asdescribed previously [Klipstein et al., Infect. Immun., 37: 550-557(1982)]. Synthetic ST was coupled to porcine immunoglobulin G (PIG) bymixing ST to PIG at a molar ratio of 100:1, using a ratio by weight of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to total conjugate proteinof 2:1, in 0.1 molar phosphate buffer, pH 7.0, for 18 hours, at 4° C.This conjugate contained 47% ST by weight and 98% ST by moles. Theconjugated ST retained 73% of its antigenicity, determined byenzyme-linked immunosorbent assay (ELISA) using hyperimmune goat andrabbit antisera to biologic ST in a previously described double sandwichtechnique [Klipstein et al., Infect. Immun., 37: 550-557 (1982)]. Theconjugate thus contained 343 antigen units (derived by multiplying thepercentage of ST present by weight times the percentage of itsantigenicity) per milligram of protein. Animals were immunizedintramuscularly with Freund's complete adjuvant (FCA) for the primaryimmunization and Freund's incomplete adjuvant for the boosterimmunization given one month later. Goats received 1,100 followed by2,300 ST antigen units, and rabbits received 300 followed by 500 Stantigen units.

Immunization and Challenge of Rats

Weanling Sprague-Dawley rats were immunized with the synthetic ST-PIGconjugate by means of an intraperitoneal primary immunization of 350 STantigen units given with Freund's complete adjuvant followed at 4 dayintervals by two peroral booster immunizations containing 700 ST antigenunits each, which were given 2 hours after the peroral administration ofcimetidine in order to ablate gastric acidity. They were challenged 5days after the final boost by the installation for 18 hours into asingle ligated ileal loop of concentrations which evoke maximumsecretion in unimmunized rats: 5 nanograms of either synthetic orbiologic ST and 0.1 milliliter of a broth culture containing 10⁹ viableorganisms per milliliter of ST-producing human E. coli strain Tx 452(078:H12). Five unimmunized and three immunized rats were challengedwith each test material. Values are expressed as the means ± standarderror of the mean percent reduced secretion in immunized rats arecompared to that in unimmunized controls; in each instance more than 50%reduced secretion represents a significant difference (P less than0.001) between the two groups as determined by Student's t-test for twoindependent means.

Antitoxin response to immunization

At the time of challenge, serum and mucosal washings were processed asdescribed previously [Klipstein et al., Infect. Immun., 37: 1086-1092(1982)] and assayed by a double sandwich ELISA in which goat hyperimmuneantiserum to synthetic ST was used for the solid phase and synthetic STwas employed as the antigen. Klipstein et al., Infect. Immun., 37:1086-1092 (1982) found that rats similarly immunized with LT, only serumantitoxin of the immunoglobulin G (IgG) and mucosal antitoxin of theimmunoglobulin A (IgA) class can be detected; therefore, antitoxin tosynthetic ST was evaluated for only these two immunoglobulin classesusing rabbit anti-rat IgG together with goat anti-rabbit antiserumconjugated to alkaline phosphatase for serum samples and goat anti-ratsecretory IgA together with rabbit anti-goat antiserum conjugated toalkaline phosphatase (Miles Research Laboratories, Elkhart, Ind.) formucosal samples. Values reported are for the geometric mean titer in 9immunized and 5 unimmunized control rats.

Section B

Enterotoxin preparations

Purified LT holotoxin was prepared by the methods described by Clementsand Finkelstein, Infect. Immun., 24: 760-769 (1979) from E. coli strain711 (F1LT), a transformed K-12 derivative bearing LT gene(s) of the Entplasmid from porcine strain P307. The B subunit was separated from theLT holotoxin by the chromatogrphic techniques described by Clements etal., Infect. Immun., 29: 91-97 (1980). The homogeneity of the LT toxinand its B subunit was confirmed by polyacrylamide gel electrophoresis asdescribed by Clements and Finkelstein, supra.

Biologic ST, obtained by growth of human E. coli strain 18D (042:H47),was purified by the methods described by Staples et al, supra, with themodification that final purification to homogeneity was achieved byelution from thin layer chromatography as described by Klipstein et al.,Infect. Immun., 37: 550-557 (1982). Synthetic ST, consisting of the samesequence of 18 amino acids described by Chan et al., supra, for pure STobtained by growth of strain 18D, was prpeared using a Beckman model 990B peptide synthesizer (Beckman Instruments Co., Irvine, CA) by methodsreported in Section II. The synthetic toxin was shown to besubstantially identical to that obtained by culture techniques (biologicST) in terms of secretory potency in the suckling mouse assay andantigenicity as determined by enzyme-linked immunoadsorbent assay(ELISA) and by seroneutralization of secretory activity in the sucklingmouse assay by hyperimmune antiserum to either the synthetic or biologictoxin in Section III, hereinbefore.

The amount of toxins used, stated as weight, was based on proteinconcentrations determined by the method of Lowry et al., supra. Molarequivalents were derived from published values of a molecular weight of91,450 daltons for LT by Clements et al., Infect. Immun., 29: 91-97(1980), 57,400 for the polymeric 5 B subunits by Gill et al., Infect.Immun., 33: 677-682 (1981), and 1,972 daltons for ST by Staples, et al.,supra.

Radioiodination of ST

Synthetic ST was radioiodinated by the chloramine-T method of Hunter,Proc. oc. Exp. Biol. Med., 133: 989-992 (1970) using proceduresdescribed previously for pure biologic ST by Klipstein et al., InfectImmun., 37: 550-557 (1982). The radiolabelled toxin contained 3×10⁵counts per minute and 71 mouse units per microgram (versus 175 mouseunits per microgram for unlabelled toxin) as determined by the sucklingmouse assay in which one mouse unit is defined as that amount whichyields an intestinal weight:carcass weight ratio of at least 0.083.

Conjugation

ST was conjugated either to LT or to the B subunit by adding1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (Sigma ChemicalCo., St. Louis, MO) to mixtures of the toxins in 0.1 molar phosphatebuffer at a pH 7.0 for 18 hours at 4° C. The conjugate was thenexhaustively dialysed against water for 48 hours at 4° C. using a 12,000molecular weight cut off dialysis bag which retained all of the LT (or Bsubunit) and conjugated ST but not unconjugated ST or EDAC. Repeateddeterminations showed that dialysis against water of either LT or the Bsubunit alone resulted in a 10% loss due to precipitation. Therefore,the amount of ST conjugated was based on the incremental increase inLowry protein present in the dialysand that was in excess of 90% of theamount of either LT or B subunit initially added. The amount ofradioiodinated ST conjugated was ascertained by comparing theradioactivity of the final conjugates to that initially added using anauto-gamma counter sold under the trademark PRIAS® PGD by PackardInstrument Co., Downers Grove, IL.

Properties of the conjugate

Unless otherwise specified, the concentration of each conjugate wasadjusted to represent 100% of the specific toxin tested in studies whichcompared the properties of conjugated toxins to those of unattenuatedtoxin. LT toxicity was compared by assaying serial 2-fold dilutions ofLT alone and in conjugated form in the Y1 adrenal cell assay of Sack etal., Infect. Immun., 11: 334-336 (1975). ST toxicity was compared byestablishing the minimal effective dosage of serial dilutions ofsynthetic ST alone or in conjugated form in the suckling mouse assay ofKlipstein et al., Infect. Immun., 37: 550-557 (1982).

Antigenicity was determined by means of ELISA. Monospecific goathyperimmune antiserum to either LT or the B subunit of cholera [Clementset al., Infect. Immun., 29: 91-97 (1980)] were used with rabbitanti-goat antiserum conjugated to alkaline phosphatase (Miles ResearchLaboratories, Elkhart, Ind.). For ST, hyperimmune antiserum to purebiologic ST raised in goats and rabbits as described previously[Klipstein et al., Infect. Immun., 37: 550-557(1982)] as used in adouble sandwich technique along with rabbit anti-goat antiserumconjugated to alkaline phosphatase; values for the conjugates werecompared to that of synthetic ST in this assay. Starting at 10micrograms, serial 2-fold dilutions were made of the conjugates andappropriate toxin. That concentration at which these preparationsyielded an absorbance of 0.600 at 410 nanometers was used to compare theantigenicity of the toxin in conjugated and unattenuated form.

Immunization procedures

Rats were given primary immunization intraperitoneally (i.p.) usingFreund's complete adjuvant followed by two peroral (p.o.) boosters at 4day intervals. Peroral immunization was given via an intragastric tube 2hours after the p.o. administration of cimetidine (sold under thetrademark TAGAMET® by Smith, Kline and French Laboratories, Carolina,Puerto Rico), at a dosage of 50 milligrams/kilogram of body weight inorder to ablate gastric secretion.

Challenge procedures

Rats were challenged 1 week after the final boost by the instillation oftest material into a single 10-centimeter ligated loop of distal ileumfor 18 hours as described previously [Klipstein et al., Infect. Immun.,31: 144-150 (1981) and 32: 1100-1104 (1981)]. Previous studies haveestablished a correlation between significant protection in this assaysystem and that achieved in immunized rats challenged by intestinalcontamination of the intact intestine [Klipstein et al., Infect. Immun.,28: 163-170 (1980)]. Challenge dosages were those which evoked maximumsecretion in unimmunized animals: 0.5 nanograms LT, 5 nanograms ofeither synthetic or biologic ST, and 0.1 milliliter of broth culturescontaining 10⁹ viable organisms per milliliter of LT⁺ /ST⁻ strain PB-258(015:H⁻), LT⁺ /ST⁺ strain H-10407 (078:H11), and LT⁻ /ST⁺ strain Tx 452(078:H12). Each datum point was determined in from 3 to 5 immunized ratsand the values reported are the mean ± standard error of the mean (SEM)degree of reduced secretion in immunized rats as compared with the valuein 5 similarly challenged unimmunized rats. Reduced secretion of 50% wassignificant for each challenge material at a P value of less than 0.001as determined by Student's t-test for two independent means.

Antitoxin response

Serum and musosal antitoxin titers to the synthetic ST and B subunitcomponents of the vaccine were determined in the serum and mucosalwashings of immunized rats by ELISA using techniques described inprevious studies which showed that the antitoxin response of ratsimmunized with LT by the parenteral/peroral approach is confined to thatassociated with serum IgG and musocal IgA [Klipstein et al., Infect.Immun., 37: 1086-1092 (1982)]. For this reason, only antitoxins of theseimmunoglobulin classes were assayed in the present study. For antitoxinto the B subunit, the B subunit was used as the solid phase; forantitoxin to synthetic ST, hyperimmune antiserum to synthetic STdeveloped in a goat was used as the solid phase and synthetic ST wasused as the antigen in a double sandwich technique. For serum samples,rabbit anti-rat IgG and goat anti-rabbit antiserum conjugated toalkaline phosphatase were added; for muscosal washings, goat anti-ratsecretory IgA and rabbit anti-goat antiserum conjugated to alkalinephosphatase (Miles Research Laboratories, Elkhart, Inc.) were used. Thevalues reported are for the increase in the reciprocal of the geometricmean titer in samples from 5 immunized over those in 5 unimmunizedcontrol rats. Antitoxin titers in control animals were 1:2 againsteither ST or B subunit in all samples except that the serum titeragainst ST was 1:4.

Section C

Preparation of the vaccine

Purified LT holotoxin was prepared from E. coli strain 711 (FILT), atransformed K-12 derivative bearing LT gene(s) of the Ent plasmid fromporcine strain P307, and separated into its subunits by chromotographictechniques as discussed above, in Section B. The homogeneity of the LTholotoxin and its B subunit was confirmed by polyacrylamide gelelectrophoresis as also discussed in Section B. Synthetic ST, consistingof the same primary structure of 18 amino acids described by Chan etal., supra, for pure ST obtained by purification of cultures of strain18D, was prepared as per Section II, supra.

The amount of toxins used was based on their protein concentrationsdetermined by the method of Lowry et al., supra; their molar equivalentswere derived from published values of a molecular weight of 57,400daltons for the polymeric form of five B subunits and 1,972 daltons forST, as discussed in Section B, above.

ST was conjugated to the B subunit by adding1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma Chemical Co., St.Louis, MO) at a ratio by weight of 1.5:1 to the total protein ofmixtures of varying molar ratios of the toxins in 0.1M phosphate bufferat pH 7.0 for 18 hours at 4° C.; the conjugate was then exhaustivelydialyzed against water and processed thereafter as described previouslyin Section B, above.

Properties of the vaccine

The antigenicity of the component toxins in the vaccine was determinedby means of enzyme-linked immunosorbent assay (ELISA) as described abovein Section B. For the B subunit, monospecific goat hyperimmune antiserumto the B subunit of human LT was used with rabbit anti-goat antiserumconjugated to alkaline phosphatase (Miles Research Laboratories,Elkhart, Ind.). For ST, hyperimmune antiserum to synthetic ST raised ingoats and rabbits (Section IV, A, above) was used in a double sandwichtechnique along with rabbit anti-goat antiserum conjugated to alkalinephosphatase. Starting at 10 micrograms, serial 2-fold dilutions weremade of the conjugates and the appropriate toxin. The concentrations atwhich the conjugate and toxin yielded an adsorbance of 0.600 at 410nanometers were compared and the value for the conjugate was expressedas a percentage of that of the unattenuated toxin. The percentageantigenicity times 1000 yielded the value for antigen units permilligram in the vaccine.

Residual toxicity in the vaccine was determined by comparing the valuesfor ST and the vaccine of (i) the minimal effective dosage in thesuckling mouse assay in which one mouse unit is defined as that amountwhich yields an intestinal weight/carcass weight ratio of at least0.083, and (ii) the ED₅₀ (that dosage which yields one-half maximumsecretion) in ligated ileal loops of unimmunized rats.

ST conjugation

In those instances where rats were immunized with synthetic ST alone,the toxin was coupled to porcine immunoglobulin G (PIG) at a molar ratioof toxin to PIG of 100:1 and a carbodiimide to total conjugate proteinratio of 1:1 by weight. This conjugate contained 46% ST by weight andhad 470 ST antigen units per milligram.

Immunization procedures

Unless specified otherwise, rats were given primary immunizationintraperitoneally (i.p.) using Freund's complete adjuvant following bytwo peroral (p.o.) boosters at 4 day intervals. Peroral immunization wasgiven via an intragastric tube 2 hours after the p.o. administration ofcimetidine (sold under the trademark TAGAMET® by Smith, Kline and FrenchLaboratories, Carolina, Puerto Rico), at a dosage of 50milligrams/kilogram body weight in order to ablate gastric secretion.

Challenge procedures

Rats were challenged 4 to 6 days after the final boost by theinstillation into a single 10-centimeter ligated loop of distal ileumfor 18 hours of 0.1 milliliters of broth cultures containing 10⁹variable organisms per milliliter. Each datum point was determined in 3to 5 immunized rats and the values reported are for the mean ± standarderror of the mean of the degree of reduced secretion in immunized ratsas compared with 5 unimmunized rats challenged with the same organisms.Reduced secretion of more than 50% is referred to as strong protectionsince it was significant in each instance at a P value of less than0.001 as determined by Student's t-test for two independent means.

Antitoxin response

Serum and mucosal antitoxin titers were determined by ELISA bytechniques described in Section B, above. For antitoxin to the Bsubunit, the B subunit was used as the solid phase; for antitoxin to ST,goat hyperimmune antiserum to synthetic ST was used as the solid phaseand synthetic ST was used as the antigen in a double sandwich technique.Since previous studies have shown that immunization with LT given by thei.p./p.o. approach arouses only serum IgG and mucosal IgA antitoxintiters [Klipstein et al., Infect. Immun., 37: 1086-1092 (1982)], onlyantitoxin of these immunoglobulin classes was evaluated. For serumsamples, rabbit anti-rat IgG and goat anti-rabbit antiserum conjugatedto alkaline phosphatase were added; for mucosal washings, goat anti-ratsecretory IgA and rabbit anti-goat antiserum conjugated to alkalinephosphatase (Miles Research Laboratories, Elkhart, Ind.) were used. Thevalues reported are for the increase in the reciprocal of the geometricmean titer in 5 immunized over that in 5 unimmunized control rats.

Section D

Preparation of the vaccine

Purified LT holotoxin was prepared from E. coli strain 711 (F1LT), atransformed K-12 derivative bearing LT gene(s) of the Ent plasmid fromporcine strain P307, and separated into its subunits by chromatographictechniques as discussed in Section B, above. The homogeneity of the LTholotoxin and its B subunit was also confirmed by polyacrylamide gelelectrophoresis as discussed in Section B. Synthetic ST, consisting ofthe same primary structure of 18 amino acids as the polypeptidedescribed by Chan et al., supra, and contained three intramolecularcystine disulfide bonds. The synthetic ST was that material whosespecific preparation was given in Section II, above. The conjugationprocedure used was the same as that discussed in Section III C, above,with the exception that the molar ratio of synthetic ST to B subunit was70:1.

Properties of the vaccine

Vaccine properties were determined as discussed in Section III C. Theconcentrations at which the vaccine and unattenuated ST and B subunityielded an absorbance of 0.600 at 410 nanometers were compared and thevalue for each component of the vaccine was expressed as a percentage ofthat of the same toxin in unattenuated form. The percentage antigenicitytimes 1000 yielded the value for antigen units (AU) per mg in thevaccine. The vaccine used contained 450 AU of each component toxin permilligram of protein, and immunization dosages described as 1000 AUcontained this amount of antigenicity for each toxin component in 2.2milligrams of vaccine.

Immunization procedures

Unless specified otherwise, rats were given primary immunizationintraperitoneally (i.p.) using Freund's complete adjuvant (FCA) followedfour days later by two p.o. boosters given at four day intervals.Peroral immunization was given via an intragastric tube 2 hours afterthe p.o. administration of cimetidine (sold under the trademark TAGAMET®by Smith, Kline and French Laboratories, Carolina, Puerto Rico), at adosage of 50 milligrams/kilogram body weight, in order to ablate gastricsecretion. When given p.o. in microsphere form, 1000 AU of the vaccinewas encapsulated by known techniques using hydroxypropyl methylcellulosephthalate (Compound HP-50, Sinetsu Chemical, Tokyo, Japan) as thepH-sensitive coating.

Rabbits were given primary immunization either by the intramuscular(i.m.) route using FCA or by the p.o. route using an intragastric tube;this was followed two weeks later by two p.o. boosters given at two weekintervals. All p.o. immunizations were preceeded 2 hours before by ani.m. injection of 30 milligrams of cimetidine.

Antitoxin response

At the time of challenge, serum and mucosal washings from challengeloops were processed [Klipstein et al., Infect. Immun., 37: 1086-1092(1982)] and antitoxin titers were determined by ELISA using reportedtechniques discussed in Section B, above. For antitoxin to the Bsubunit, the B subunit was used as the solid phase; for antitoxin to ST,goat hyperimmune antiserum to synthetic ST was used as the solid phaseand synthetic ST was used as the antigen in a double sandwich technique.Since previous studies, using LT as the antigen, have shown thatimmunization by the i.p./p.o. approach arouses only serum immunoglobulinG(IgG) and mucosal IgA antitoxin titers [Klipstein et al., Infect.Immun., 37: 1086-1092 (1982)], only antitoxin of these immunoglobulinclasses were evaluated. The values reported are for the increase in thereciprocal of the geometric mean titer in four or more immunized animalsover that in five unimmunized control animals, except for rabbit serawhere pre- and post-immunization samples from the same animal werecompared.

Challenge procedures

Immunized animals were challenged four to seven days after the finalbooster immunization by the instillation of graded dosages of either STor LT into ligated ileal loops for 18 hours as described previously[Klipstein et al., Infect. Immun., 31: 144-150 (1981); Sack, Infect.Immun., 8: 641-648 (1973)]. The toxin was instilled in 0.5 millilitersof normal saline into a single loop in each rat and in 1.0 millilitersof Trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD) inup to 10 loops in each rabbit. The values presented for each datum pointof fluid secretion are the mean ± standard error of the mean (SEM) infrom three to five control and immunized rats and in loops in sixcontrol rabbits and four rabbits in each immunization group. Theprotection index (PI) was determined by dividing that dosage of toxin inimmunized animals which yielded the same secretion as the 50% effectivedose (ED₅₀) in unimmunized animals by the value for unimmunized animals.

Rats were also challenged with 0.1 milliliter of a broth culturecontaining 10⁹ viable organisms of LT⁻ /ST⁺ strain Tx 452 (078:H12) permilliliter. The results are expressed as the mean ± SEM percentagereduced secretion in immunized rats as compared to the value in fivesimilarly challenged unimmunized control animals. The statisticaldifference between secretion in the immumized and control groups wasdetermined by Student's t-test for two independent means.

V. USAGE OR MULTIMERIC ST

Multimeric ST molecules prepared as discussed hereinbefore were linkedwith a carrier molecule in determinations also carried out under thedirection of Dr. Frederick A. Klipstein, supra. The results of thosedeterminations are discussed hereinbelow.

A. Vaccines Containing Monomeric And Multimeric ST Molecules

Three vaccines were prepared. The first contained monomeric synthetic ST(hereinafter sometimes referred as M-ST), the second contained theheat-to-tail multimeric dimer ST sometimes referred herein as ST/ST,while the third vaccine contained the polymeric ST whose repeating unitsare linked together by intramolecular, interpolypeptide cystinedisulfide bonds that is sometimes referred to as P-ST. The synthesis ofeach of the above materials has been described in Section IIhereinbefore. Each of the ST molecules was bonded to the B subunit of E.coli heat-labile toxin (LT) as is discussed hereinafter in Section VI A.

Properties of Synthetic ST Preparations

When tested by the ELISA technique discussed hereinbefore usinghyperimmune antisera to an M-ST conjugate, the antigenicity ofnon-conjugated ST/ST was 3.5-fold greater and the antigenicity ofnon-conjugated P-ST was 15-fold greater than that of non-conjugatedM-ST. The results of these antigenicities for these determinations areillustrated in the graphs of FIG. 19.

When graded amounts of each ST preparation were tested in the sucklingmouse assay, supra, values for one mouse unit were 5.7 nanograms forM-ST, 45 nanograms for ST/ST and 18.2 nanograms for P-ST. Thus, the freeST/ST had 13 percent of the secretory potency of M-ST, while P-ST had 31percent of the secretory potency of M-ST.

Conjugation with the B Subunit

Each of the three synthetic ST preparations was cross-linked to the LT Bsubunit using varying initial molar ratio of ST to B subunit. Theantigenicities of the conjugates so prepared are illustrated in thethree graphs in FIG. 20 as a function of toxin percentage by weight andalso of the initial molar ratio of the two toxins in the conjugate. Itis to be understood that "cross-linking" of a toxin to a carrier isdifferent from "cross-linking" of a polypeptide by interpolypeptidecystine disulfide bonds.

Conjugates that contained equal portions by weight of ST and B subunitswere obtained from reactions in which the intial molar ratios of ST to Bsubunit were 45:1 for M-ST and 40:1 for ST/ST. Sufficiently large ratiosof P-ST to B subunit were not utilized to yield such a conjugate.

The antigenicity of the toxins was not affected by the cross-linkingreaction so that the M-ST--B subunit conjugate that contained equalproportions of each toxin component by weight had 500 antigen units ofeach component. In contrast, conjugates of the hyperantigenic STpreparations (ST/ST and P-ST) with approximately equal proportions ofantigen units of each toxin component contained only 25 percent of ST/STand 9 percent of P-ST by weight.

Since the proportion of weight of B subunit became reciprocally greateras the weight of ST decreased, the amount of B antigen units wasgreatest in those conjugates containing the least amount of ST. Amongconjugates containing equal proportions of antigen units of each toxincomponent, the M-ST vaccine had 500 antigen units per milligram, theST/ST vaccine contained 725 antigen units per milligram and the P-STvaccine had 900 antigen units per milligram of each toxin component.

The residual toxicity of vaccines made from the two multimeric SToperations was 10-fold less than that of the vaccine made with M-ST.This was attributable principally to the fact that these vaccinescontained smaller amounts of ST.

Although the multimeric ST preparations were less toxic initially thanM-ST, their toxicities were also less strongly attenuated by thecross-linking conjugating reaction. Thus, the toxicity of each STpreparation was compared to its original value; the toxicity of M-ST wasreduced 727-fold by the cross-linking reaction, that of P-ST was reducedby 256-fold and that of ST/ST was reduced by only 44-fold. Several ofthe properties of these vaccines are shown in Table 9 hereinbelow.

                  TABLE 9                                                         ______________________________________                                        Properties Of Cross-Linked Vaccines Obtained                                  Using The B Subunit And Different                                             Synthetic ST Preparations                                                     Conjugation                                                                   Reaction      Vaccine       1000 AU Vaccine                                          Molar.sup.b                                                                          Wt. %.sup.c                                                                           AU/mg.sup.d                                                                             Amount  Tox.sup.g                             ST.sup.a ST:B     ST    B   ST   B    Vac.sup.e                                                                          ST.sup.f                                                                           ST                            ______________________________________                                        M-ST     45:1(0.5)                                                                              52    48   510 500  2.00 1040 900                           ST/ST    20:1(0.7)                                                                              25    75   849 737  1.38  345  97                           P-ST     10:1(0.9)                                                                               9    91  1300 917  1.11  100 108                           ______________________________________                                         .sup.a ST preparations in the conjugate. MST = monomeric synthetic ST;        ST/ST = headto-tail dimer synthetic multimeric ST; and PST = polymeric ST     containing interpolypeptide cystine disulfide bonds.                          .sup.b Initial molar ratio of ST preparation to LT B subunit.                 Parenthesized numbers denote the weight ratio of glutaraldehyde to total      protein in the reaction.                                                      .sup.c Relative weight percentage of each toxin in each conjugate.            .sup.d Antigen units (AU) of each toxin per milligram of vaccine              conjugate.                                                                    .sup.e Amount of vaccine conjugate in milligrams needed to provide 1000 A     of each toxin component.                                                      .sup.f Amount of ST in micrograms contained in a vaccine conjugate            providing 1000 AU of each toxin.                                              .sup.g Equivalent amount of unattenuated ST secretory potency in nanogram     as determined by the suckling mouse assay in a vaccine conjugate              containing 1000 AU of each toxin.                                        

Immunization of Rats With ST--B Subunit Vaccines

Groups of rats were immunized with two vaccines: (i) M-ST cross-linkedat an initial molar ratio of 70:1 to B subunit by the carbodiimidereaction as described hereinbefore, and in Section IV B to provide aconjugate that contained 51 percent M-ST and 49 percent B subunit byweight, and that had 450 antigen units of each toxin component permilligram; and (ii) P-ST cross-linked at an initial molar ratio of 10:1to B subunit by the glutaraldehyde reaction (discussed in Section VI A)to provide a conjugate that contained 9 percent P-ST and 91 percent Bsubunit by weight, and that contained 1300 M-ST and 917 B subunitantigen units per milligram. The dosages used were based upon the amountof M-ST antigen units per milligram of each vaccine. All rats receivedperenteral primary immunization with 200 MST antigen units and varyingdosages of peroral (p.o.) booster immunizations such that total p.o.dosages ranged between 500 and 3000 M-ST antigen units.

The response to the M-ST and P-ST conjugate-containing vaccines wasidentical when the total p.o. immunization total was expressed in M-STantigen units as can be seen by an examination of FIG. 21A. Whenexpressed as total p.o. immunizations, each vaccine raised a similardose-dependent increase in the mucosal IgA M-ST anti-toxin response, anda similar degree of protection against a challenge with a viable LT⁻/ST⁺ of E. Coli was observed.

There was a striking difference, however, when the p.o. dosages wereexpressed on the basis of the amount of ST given by weight. Thus,examination of FIG. 21B illustrates that P-ST was markedly moreeffective; i.e., the p.o. dosage of conjugated P-ST necessary to yield50 percent reduced secretion in challenged rats was 70 micrograms, whichis 15-times less than the 1050 micrograms of conjugated M-ST needed toachieve that result.

The above results shown in Table 9 and in FIG. 21 illustrate severalfeatures of the present invention. First, both of the multimeric STpreparations had an improved antigenicity per weight over monomeric ST.In addition, both preparations showed reduced toxicity in the sucklingmouse assay as free molecules and as conjugates with the LT B subunit.The highly antigenic P-ST preparation yielded a vaccine with greaterantigenic potency but with substantially identical toxicity to thevaccine derived from the ST/ST. That P-ST--B subunit vaccine had nearlytwice the antigenic potency of both toxin components (ST and B subunit)but only about one tenth of the residual ST toxicity of the M-ST--Bsubunit vaccine. Still further, the amount (380 micrograms) of P-STneeded to make a vaccine containing 1000 antigen units of each toxincomponent was about one-quarter of the amount (1490 micrograms) of theM-ST necessary to make such a vaccine.

It is also important to note that the antigenicity of the multimeric STpreparations as determined by ELISA correlated with their immunogenicityas determined by their effectiveness in arousing an anti-toxin responseand in providing protection against the challenge in immunizedexperimental animals. Since the antigenicity of ST in conjugates is afunction both of the proportion of ST in that conjugate and of thedegree to which its antigenicity was compromised by the cross-linkingconjugating reaction, expression of the dosage of the conjugate on aweight basis does not provide meaningful information unless theantigenicity is directly measured. When the antigenicity of ST isdetermined by a direct assay of the conjugate using an ELISA technique,and expressed in antigen units, then immunization with M-ST conjugatedeither to the B subunit or to an immunologically non-specific proteincarrier, yields a dose-dependent response of mucosal anti-toxin levelsand a degree of protection against challenge with viable toxin-producedstrains Klipstein et al., Infect. Immun., 40: 924-929 (1983).

The above findings further show that immunization with either the M-STor the P-ST vaccine raised an identical response when dosages wereexpressed as M-ST antigen units. Those observations also confirmed thecorrelation between antigenicity and immunogenicity in that P-ST was 15times more antigenic than M-ST as determined by ELISA, and 15 times moreimmunogenic by weight than was M-ST in immunized rats.

The above studies also incorporated two modifications not present inpreviously published reports. The first was that the B subunit wasobtained directly from an E. coli K-12 strain that had been modified byrecombinant techniques to produce only human B subunit, rather thanobtaining that subunit by dissociation procedures from the porcine LTholotoxin as has been done in the past. The second modification was thatglutaraldehyde was substituted for a carbodiimide as a cross-linkingreagent.

Coupling using glutaraldehyde has a number of advantages compared tocouplings using carbodiimides. First, it provides effectivecross-linking of ST to the B subunit without affecting the antigenicityof either toxin. Second, it yields more efficient coupling of ST to Bsubunit so that vaccines containing equal antigenic proportions of STand B subunit can be derived from lower initial molar ratios of ST to Bsubunit, thereby significantly reducing the amount of ST needed to makethe vaccine. Third, unlike carbodiimides, there is considerableexperience that attests to the safety of administering glutaraldehydeconjugates to humans. [See, Cockroft et al., J. Allerg. Clin. Immunol.,60: 56-62 (1977); Levine et al., Infect. Immun., 21: 158-162 (1978); andRelyveld, Prog. Clin. Biol. Res., 47: 51-76 (1980).]

B. Further Vaccines Containing Monomeric And Polymeric ST Molecules

Further conjugates of two of the before-described three synthetic STpreparations, M-ST, and P-ST were prepared with the LT B subunit. Theeffects of differing initial molar ratios of the ST to B subunit, basedon the molecular weight of monomeric synthetic ST (M-ST) were examinedas were different coupling agents. The coupling agents utilized in thisstudy were 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC),dimethyl suberimidate (DMS) and glutaraldehyde (GA).

Results from some of these studies are shown in the graphs of FIG. 22for P-ST coupled with either DMS or EDAC. The results using GA are shownin the right-hand panel of FIG. 20 (panel C). As can be seen, both DMCand GA provide greater ST antigenicities (antigen units per milligram)at lower initial molar ratios of the P-ST to B subunit than are obtainedusing EDAC. Data for particular conjugates are shown in Table 10, below.

                                      TABLE 10                                    __________________________________________________________________________    Properties of Vaccines Prepared From The B Subunit And                        Different ST Preparations With Differing Coupling Agents                      Conjugation  Vaccine   1000 AU Vaccine                                        Reaction     Molar.sup.c                                                                       Wt. %.sup.d                                                                         AU/mg.sup.e                                                                           Amount  Tox.sup.h                              ST.sup.a                                                                           Coupler.sup.b                                                                         ST:B                                                                              ST  B ST   B  Vac.sup.f                                                                          ST.sup.g                                                                         ST                                     __________________________________________________________________________    M-ST EDAC(1.5)                                                                             70:1                                                                              51  49                                                                              492  467                                                                              2.20 1122                                                                             1276                                   P-ST EDAC(1.5)                                                                             30:1                                                                              36  64                                                                              651  687                                                                              1.51 540                                                                              332                                    M-ST DMS(1.0)                                                                              50:1                                                                              46  54                                                                              455  508                                                                              2.20 1012                                                                             770                                    P-ST DMS(1.0)                                                                              10:1                                                                              12  88                                                                              1620 780                                                                              1.33 160                                                                              172                                    M-ST GA(0.5) 45:1                                                                              52  48                                                                              510  500                                                                              2.00 1040                                                                             900                                    P-ST GA(0.6) 30:1                                                                              30  70                                                                              4130 679                                                                              0.25 75 92                                     __________________________________________________________________________     .sup.a ST preparations used in the conjugate. MST = monomeric synthetic       ST; and PST = polymeric ST containing interpolypeptide cystine disulfide      bonds.                                                                        .sup.b Coupling agents utilized to prepare the conjugate. EDAC =              1ethyl-3-(3-dimethylaminopropyl) carbodiimide; DMS = dimethyl                 suberimidate; and GA = glutaraldehyde. Parenthesized numbers indicate the     ratio by weight of coupling agent to total initial protein present in the     coupling reaction medium.                                                     .sup.c Initial molar ratio of ST preparation to LT B subunit.                 .sup.d Relative weight of each toxin in each conjugate.                       .sup.e Antigen units (AU) of each toxin per milligram of vaccine              conjugate.                                                                    .sup.f Amount of vaccine conjugate in milligrams needed to provide 1000 A     of each toxin component.                                                      .sup.g Amount of ST by weight in micrograms contained in a vaccine            conjugate providing 1000 AU of toxin.                                         .sup.h Equivalent amount of unattenuated ST secretory potency in nanogram     in a vaccine conjugate containing 1000 AU of ST toxin as determined by th     suckling mouse assay.                                                    

The above data illustrate the superiority of polymeric synthetic ST(P-ST) over the monomeric synthetic ST (M-ST) on a per weight basis.That superiority does not appear to be dependent upon the coupling agentused, although the different coupling agents do show some effects. Thus,glutaraldehyde (GA) and dimethyl suberimidate (DMS) appear to providesuperior conjugates of both P-ST and M-ST to the B subunit as comparedto the carbodiimide (EDAC).

Experimental details regarding the above studies, in addition to thosealready provided, are found in Section VI B, hereinafter.

C. Immunogenicity of Non-Conjugated Polymeric ST

Each of the above-described ST preparations has been shown to beantigenic, and each is defined as having at least 10 percent of theantigenicity of the native, biologic ST on a weight basis. Thoseantigenicities are illustrated by ELISA determinations. In addition, theabove ST preparations when coupled to a protein carrier such as the LT Bsubunit or porcine immunolglobulin G have been shown to be immunogenicin both rats and in rabbits. However, as far as is known, the monomericST (M-ST) preparations are haptenic, and are not immunogenic when usedalone without a coupled carrier.

Immunization with ST as a conjugate with a carrier protein whose carrieris not an active immunogen is wasteful of resources since a portion ofthe conjugate is not immunologically active. The B subunit portion ofST--B subunit conjugates is of course partly immunogenic and does inducethe production of antibodies. However, sufficient quantities of theholotoxin or of the B unit are not readly available to provide aneconomically feasible vaccine, and when produced by genetic engineeringtechniques, preparations of such carrier proteins may also containunwanted protein impurities.

It would therefore be of great benefit if a totally synthetic immunogencould be prepared for a vaccine in which substantially the entireconjugate were immunologically active. The polymeric ST preparation,P-ST, because of its high average molecular weight, solubility,antigenicity and sucessful use as a coupled immunogen in a conjugateappeared to be such an immunogenically active carrier for a syntheticpolypeptide corresponding in amino acid residue sequence to animmunogenic, and antigenic determinant region of the LT B subunit, suchas those sequence noted in Table 2, supra. Immunogenicity studies weretherefore undertaken using non-conjugated P-ST preparation as a firststep toward the production of such a P-ST--LT B polypeptide conjugate.

Rabbits were immunized with non-conjugated P-ST, followed by two boostswith P-ST. ELISA determinations revealed that sera obtained from thoseimmunizations had high titers against synthetic monomeric ST (M-ST) asan antigen. Indeed, a serum titer of 1:2500 provided one-half of themaximal saturation of the M-ST antigen.

The titers obtained are sufficient to indicate that a carrier is notneeded to immunize and protect against challenge with non-conjugatedP-ST. Those titers are also sufficient to suggest that P-ST can itselfbe used as a carrier for conjugation of an immunogenic, synthetic LT Bsubunit polypeptide to provide a totally synthetic immunogen,substantially all of which is immunogically active, and that providesprotection against both ST- and LT-producing pathogens. It is believedthat M-ST administered under the same protocol used in these studies;i.e., non-conjugated to a carrier, would induce substantially no anti-STantibodies.

The procedures used to obtain these results are discussed in Section VIC, hereinafter.

VI. MATERIALS AND METHODS FOR MULTIMERIC ST A. Vaccines Containing M-ST,ST/ST and P-ST Enterotoxin Preparations

Three synthetically-produced ST preparations were used. The monomericsynthetic ST (M-ST) utilized in these preparations is that material thatcontains the same sequence of 18 amino acid residues as that describedfor natural human ST Ib by Chan et al., J. Biol. Chem., 256: 7744-7746(1981), whose biologic and antigenic properties are substantiallyidentical to those of native ST. The preparation and properties of M-SThave been discussed in Sections II and III hereinbefore. The ST/STpreparation utilized is the 36 amino acid-containing polypeptide that isthe head-to-tail dimer of the 18 amino acid residue-containing human STIb whose preparation was discussed hereinbefore in Section II. Thepolymeric ST (P-ST) is that material whose plurality of ST repeatingunits are linked by intramolecular, interpolypeptides cystine disulfidebonds. The preparation of P-ST has also been described hereinbefore inSection II. The ST/ST and P-ST preparations were purified by passagethrough a Sephadex G-50 chromagotraphy column, lyophilized, and werethen used without further purification.

The B subunit was obtained from cultures of E. coli strain pDF 87 atransformed K-12 derivative bearing the B subunit plasmid of human E.coli strain H 10407 [Clement et al., Infect. Immun., 40: 653-658(1983)], and was purified by the chromatographic techniques described inClements et al., Infect. Immun., 29: 91-97 (1980). The amount of toxinsused was based upon their protein concentrations determined by themethod of Lowry et al., J. Biol. Chem., 193: 265-275 (1951); the molarequivalents were derived from published values for the native B subunit(57,400 daltons for the polymeric five subunits) and native ST (1,972daltons), as discussed hereinbefore and in Gill et al., Infect. Immun.,33: 677-682 (1981) and Staples et al., J. Biol. Chem., 255: 4716-4721(1980), respectively.

Conjugation Conditions

Three different synthetic ST preparations were cross-linked to LT Bsubunit by mixing varying initial molar ratios of those toxins in thepresence of Sigma grade I glutaraldehyde (GA) (Sigma Chemical Co., St.Louis, MO). The ratio of GA to total protein of the toxin mixture wasvaried in each reaction so that the GA to B subunit ratio was keptconstant at a 700:1 molar ratio. This concentration of GA has been foundto provide effective cross-linking without attenuation of B subunitantigenicity. Following a two hour reaction at room temperature, theconjugates were exhaustively dialyzed against TEAN buffer (Tris, EDTA,sodium azide, sodium chloride) at four degrees C. and then processed asdescribed hereinbefore and in Klipstein et al., J. Infect. Dis., 147:318-326 (1983).

Properties of the Conjugates

The antigenicity of the synthetic ST preparations, either alone or inconjugated form, was determined by a double sandwich enzyme-linkedimmunosorbant assay (ELISA) using goat and rabbit hyperimmune antiserasynthetic M-ST as described in Klipstein et al., J. Infect. Dis., 147:318-326 (1983) and Klipstein et al., Infect. Immun., 39: 117-121 (1983).The antigenicity of the B subunit in the conjugates was determined byELISA using goat hyperimmune antiserum to human B subunits. Theantigenicity of each toxin component of the conjugate is expressed as apercentage of that of the concomitantly-assayed respective unattenuatedtoxin; the percent antigenicity of each toxin component times 1000yielded the number of its antigen units per milligram of vaccine. Theantigenicity of the two multimeric ST preparations in conjugates isexpressed in terms of M-ST antigen units.

Residual ST toxicity of the vaccines was determined as describedpreviously by Klipstein et al., Infect. Immun., 37: 550-557 (1982) bycomparing the values for unattenuated ST. For each vaccine, the minimaleffective dosage was determined in the suckling mouse assay, in whichone mouse unit is defined as that amount which yields an intestinalweight to carcass weight ratio of at least 0.083.

Immunization Procedures

150-175 Gram Sprague-Dawley rats were given primary immunizationintraperitoneally (i.p.) using complete Fruend's adjuvant followed bytwo boosters given perorally (p.o.) at 4-day intervals. Immunizationp.o. was given via an intragastric tube two hours after the p.o.administration of cimetidine (TAGAMET® available from Smith Kline &French Laboratories, Carolina, Puerto Rico) at a dosage of 50 milligramsper kilogram of body weight to ablate gastric secretion.

Challenge Procedures

The rats were challenged four to six days after the final booster by theinstillation of 0.1 milliliters of a broth culture containing 10⁹ viableorganisms per millilter of E. coli LT⁻ /ST⁺ strain Tx 452 (O78: H12) ina single 10-centimeter ligated loop of distial ileum for 18 hours, asdescribed hereinbefore and in Klipstein et al., Infect. Immun., 34:637-639 (1981) and in Klipstein et al., Infect. Immun., 40: 924-929(1983). Each datum point was determined in four to six immunized rats,and the results reported are for the mean (plus or minus the standarderror of the mean) percentage of reduced secretion in immunized rats ascompared with the value in five unimmunized control rats that weresimilarly challenged. A reduced secretion of greater than 50 percentrepresented a significant (P less than 0.001) difference, as determinedStudent's t test for two independent means, between values in immunizedand control animals.

Anti-Toxin Response

Mucosal IgA anti-toxin (mucosal AT) titers to M-ST and to the B subunitwere determined by ELISA as described hereinbefore, and in Klipstein etal., Infect. Immun., 37: 550-557 (1982) and in Klipstein et al., Infect.Immun., 40: 924-929 (1983). Anti-toxin titers in animals immunized withP-ST vaccine were determined by a double sandwich technique in whichgoat hyperimmune antiserum to M-ST was used as the solid phase and M-STwas the antigen. Those results are expressed as M-ST values. The valuesreported are mean fold increases, rounded to the nearest integer value,in the titers of immunized rats over those of in unimmunized controlrats. Anti-toxin titers in the control rats were 1:2 against both toxincomponents; thus, a titer of 1:64 in immunized animals represented6-fold increase.

B. Further Vaccine Conjugates Containing Monomeric And Polymeric STMolecules

The monomeric synthetic ST (M-ST) and the polymeric synthetic ST (P-ST)preparations used in these conjugates were those whose preparations anduses have been discussed hereinbefore in Sections II, III and VI A. TheLT B subunit was the material discussed in Section VI A.

Coupling of M-ST and P-ST to the B subunit using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) was carried out asdiscussed in Section IV for M-ST. Thus, the amounts of both M-ST andP-ST used were based upon both materials being monomeric.

Couplings using glutaraldehyde (GA) were carried out as described inSection VI A with the exception that the amount of GA utilized isexpressed as a weight ratio of GA to total protein of the conjugate of0.5:1 or 0.6:1, rather than the 700:1 molar ratio of GA to B subunitutilized in Section VI A.

Couplings using dimethyl suberimidate (DMS) were conducted at a DMS tototal protein weight ratio of 1:1. Exemplary procedures for suchcouplings can be found in Carpenter et al., J. Biol. Chem., 247:5580-5586 (1972) and in Hillel et al., Biochemistry, 16: 3334-3342(1977).

Antigenicities of the conjugates, the number of antigen units (AU) permilligram of conjugate, the amount of conjugate-containing vaccine touse to provide 1000 AU of the toxins and the amount of ST preparation insuch a vaccine were determined as discussed in Section VI A. Thesecretory potency of the conjugates was determined using thebefore-described suckling mouse assay.

C. Immunogenicity of Non-Conjugated Polymeric ST

The polymeric synthetic ST (P-ST) used in these determinations was thematerial purified as discussed hereinbefore in Sections II and VI A. Theimmunization and ELISA protocols utilized are as follows.

Rabbits were injected intramuscularly (i.m.) on day zero with a vaccinethat contained 200 micrograms of P-ST dispersed in 1.5 milliliters ofcomplete Freund's adjuvant (CFA) and 1.0 milliliter ofphosphate-buffered saline (PBS) having a pH value of 7.2. The animalsreceived booster intraperitoneal (i.p.) injections of a vaccine thatcontained the same amount of P-ST contained in a 1.0 milliliters ofincomplete Freund's adjuvant plus 1.0 milliliters of PBS on day 14,followed by second booster injections of a vaccine that contained thesame amount of P-ST sorbed onto alum, plus 1.5 milliliters of PBS on day27. Serum samples from the immunized rabbits were obtained by ear veinon day 34 after the first immunization.

The 18-mer monomeric synthetic ST (M-ST) whose synthesis and propertiesare discussed in Sections II, III and IV, and that had the amino acidresidue sequence of ST Ib, supra, was used as the antigen in ELISAassays for antiserum titers. The antigen was dissolved in PBS at pH 7.2to provide a stock solution containing 1 milligram per milliliter. Thatstock solution was then diluted with a 0.1 molar sodium acetate bufferhaving a pH value of 9.6 to provide a second stock solution containingM-ST at a concentration of 0.5 micrograms per milliliter.

50 Microliters of the second stock solution were placed into the wellsof a microtiter plate, to provide 25 nanograms of the M-ST antigen toeach well. The plate was then incubated in a moist chamber for a periodof 2 hours at a temperature of 37° C.

The wells were then washed three times with a PBS solution thatcontained 0.07 percent Polysorbate 20 (TWEEN® 20; ICI United States,Inc., Wilmington, DE), followed by two washes with distilled water. 100Microliters of 1 percent bovine serum albumin (BSA) in PBS were thenadded to each well to block sites of non-specific binding, and theBSA-containing solutions were incubated in the wells for 1 hour at 37°C.

Solutions containing unbound BSA were shaken from the plate. Withoutfirst drying the plate, 90 microliters of PBS were added to each well ofthe top row of wells, while 50 microliters of PBS were added to eachwell of the remainder of the plate. Thereafter, 10 microliters ofanitserum were added to each of the top row of wells, and the admixtureso made was mixed. 50 Microliters of antiserum-containing solution fromeach well was then added and mixed with the 50 microliters of PBS in thewell therebelow to provide further two-fold dilutions. Similar serialtwo-fold dilutions were continued for the remaining wells of the plate.After all of the dilutions had been made, the dilutedantiserum-containing wells were incubated at 37° C. for a period of 1hour.

Goat anti-rabbit serum labeled with peroxidase was diluted 1:3000 byvolume with PBS also containing 1 percent BSA, and 50 microliters of theresulting solution was added to each well, except for the wells of thetop row that were not analyzed. The wells so prepared were incubated fora period of 1 hour at 37° C., and were then washed as described above.

A solution of ortho-phenylenediamine (OPD) was prepared by dissolvingone tablet of OPD Substrate (Pittman-Moore, Inc., Washington Crossing,NJ) in 6 milliliters of distilled water to which one drop of 3 percenthydrogen peroxide was also added. 80 Microliters of the resultingsolution were added to each of the microtiter plate wells. Colordevelopment in the wells was stopped after 20 minutes by the addition of50 microliters of 4 normal sulfuric acid to each well. The amount ofcolor present in each well was determined by measuring the opticaldensity of the liquid therein at 492 nanometers.

The amount of antibody in the rabbit serum required to saturate one-halfof the M-ST antigen on the plate was thereafter calculated usingstandard techniques. It was found as a result of those calculations thatthere was sufficient antibody to M-ST present in the immunized rabbitserum to saturate one-half of the applied M-ST antigen when that serumwas diluted about 1:2500. Thus, an antiserum titer of 1:2500 wasobtained by this double-sandwich ELISA determination.

VII. DIAGNOSTICS

The results discussed hereinbefore amply illustrate that the syntheticST preparations of this invention are antigenic. For example, themonomeric synthetic ST has been shown to have an antigenicity of about40 to about 260 percent of biologic ST when assayed using antibodies tobiologic ST, and an antigenicity of about 10 to about 100 percent whenmeasured using antibodies raised to a particular synthetic STpreparation; i.e., the purified product of preparation (3) of Table 2.Similarly, the multimeric dimer ST/ST has been shown to have anantigenicity that is about 300-350 percent of that of monomeric ST(M-ST), while the polymeric ST (P-ST) has been shown to have about900-1500 percent the antigenicity of M-ST.

The synthetic ST molecules of this invention, and particularly themultimeric ST forms, can therefore be useful as diagnostic reagents oras part of a diagnostic system used to identify animals, includinghumans, that are infected with bacteria such as E. coli and Klebsiellapneumoniae that product ST toxins. Antibodies raised to the ST moleculesof this invention may also be useful in such diagnostics.

Immunoassays are particularly preferred diagnostic methods of utilizingST molecules. The ELISA determinations already discussed are exemplaryof such immunoassays. Additionally, useful immunoasays includeradioimmune assays and fluorescence immune assays, and the like.

One embodiment of this diagnostic system invention is particularlyuseful in competition assays and includes a first reagent and a secondreagent in separate containers. A first reagent comprises a syntheticantigenic ST polypeptide of this invention such as M-ST or P-ST as theantigen. The second reagent comprises receptors such as antibodies thatimmunoreact with M-ST or P-ST and also immunoreact with biologic ST suchas those discussed hereinbefore. A means for indicating the presence ofan immunoreaction between the antigen and receptors is signalled byfurther, anti-receptor, receptors linked to a tag such as a radioactiveelement like ¹²⁵ I, a fluorescent dye like fluorescein or an enyzme likeperoxidase. The indicating means is included either in a separatecontainer as in phosphatase-linked goat-antirabbit antibodies withanother separate container for its substrate, or along with theantibodies as where radioactive elements are bonded to the antibodies.The indicating means can also be separately supplied.

Admixture of predetermined amounts of the first and second reagents inthe presence of a predetermined amount of a sample to be assayed such asa stool sample or a bacterial culture from a stool sample provides anamount of immunoreaction signalled by the indicating means. The amountof the immunoreaction is different from a known amount of immunoreactionwhen an ST is present in the assayed sample.

In usual practice, the bacterial culture is pre-incubated with theantibody and that composition is then incubated with the P-ST that isbound to the walls of an ELISA well. Rinsing of the well to remove anyantibody-natural, biologic ST complex (immunoreactant) leaves an immunecomplex of the P-ST and antibody whose presence and amount may besignalled by the indicating means.

The use of whole, intact, biologically active antibodies is notnecessary in many diagnostic systems such as the competition assaydiscussed immediately above. Rather, only the biologically activeidiotype-containing amide portion of the antibody molecule that binds tothe antigenic ST may be needed. Illustrative of the iodiotype-containingpolyamide portions are those known as Fab and F(ab')₂ antibody portionsthat are prepared by well-known enzymatic reactions on typically wholeantibodies.

Whole, intact antibodies, Fab, F(ab')₂ portions and the like thatcontain the antibodies' idiotypic regions are denominated herein asreceptors. The phrase "receptor" is used above and in the appendedclaims to embrace the group of such molecules as are useful indiagnostic products or techniques. However, while Fab or F(ab')₂antibody portions may be utilized as the receptor of a diagnostictechnique or product, use of the whole, intact antibody is usuallypreferred, if only because preparation of an Fab or F(ab')₂ portion ofan antibody requires additional reaction and purification of sera.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the novel conceptsof the invention.

What is claimed is:
 1. An antigenic synthetic polypeptide multimercomprising a plurality of polypeptide repeating units bonded together(1) head-to-tail through an amide bond formed between the amine group ofthe amino-terminal residue of a first polypeptide repeating unit and thecarboxyl group of the carboxy-terminal residue of a second polypeptiderepeating unit, or (2) by intramolecular interpolypeptide cystinedisulfide bonds formed between Cys residues of said polypeptiderepeating units, said synthetic multimer having at least about 10percent of the antigenicity of that of biologic heat-stable enterotoxinof Escherichia coli and capable of inducing antibodies to saidenterotoxin, and having thin layer chromatographic and electrophoreticmobilities different from said biologic heat-stable enterotoxin, saidrepeating units including the amino acid residue sequence, taken fromleft to right and in the direction from amino-terminus tocarboxy-terminus, represented by the formula: ##STR15## wherein thethree specific amino acid residues in parentheses are each analternative to the immediately preceding amino acid residue in saidsequence;a-f and g-l are integers each having a value of zero or one,with the proviso that if the value of any of a-f or g-l is zero, thecorresponding R_(a-f) ¹⁻⁶ - or R_(g-l) ⁷⁻¹² -group is absent, and whenan R_(a-f) ¹⁻⁶ -group is absent the sulfur atom of the respective Cysresidues having an absent R_(a-f) ¹⁻⁶ -group forms a cystine disulfidebond, while if the value of said a-f or g-l is one, said correspondingR_(a-f) ¹⁻⁶ - or R_(g-l) ⁷⁻¹² -group is present; said R_(a-f) ¹⁻⁶-groups when taken individually, are the same or different moietiesbonded to the sulfur atom of the respective Cys residues of the formulaand are selected from the group consisting of hydrogen, an alkyl groupcontaining 1 to about 4 carbon atoms, and a substituted alkyl groupcontaining 2 to about 20 carbon atoms; said R_(g-l) ⁷⁻¹² -groups arealternative Ser amino acid residues to each immediately preceding Cysresidue shown in the formula; at least two of a-f and two of g-l arezero and two Cys residues are present with the proviso that saidsynthetic polypeptide contains at least one intramolecular cystinedisulfide bond formed from the at least two Cys residues present; and"m" is an integer having the value of zero or one with the proviso thatif "m" is zero R_(m) ¹³ is absent, and if "m" is one, R_(m) ¹³ is alinking group, the acyl portion of a carboxylic acid containing 1 toabout 20 carbon atoms that forms an amide bond with the amine of theamino-terminal residue, an amino acid residue, or a polypeptide chain ofamino acid residues wherein the amino acid residues of said polypeptideare taken from left to right and in the direction from amino-terminus tocarboxy-terminus, and selected from the group consisting of(a) Tyr; (b)Asn(Ser)Thr(Ser)Phe(Asn)Tyr; (c) ##STR16## (d) ##STR17## (e) ##STR18##(f)MetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsn(Ser)Thr(Ser)Phe(Asn)Tyr;(g) TyrThrGluSerMetAlaGlyLysArgGlyAsn(Ser)Thr(Ser)Phe(Asn)Tyr; (h)TyrThrGluSerMetAlaGlyLysArgGluMetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsn(Ser)Thr(Ser)Phe(Asn)Tyr;(i)MetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLys;(j) TyrThrGluSerMetAlaGlyLysArgGly; and (k)TyrThrGluSerMetAlaGlyLysArgGluMetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLyswherein the immediately above parenthesized amino acid residues R_(a-f)¹⁻⁶ and R_(g-l) ⁷⁻¹² are as before defined; said polypeptide having atleast about 10% of the antigenicity of that of biologic heat-stableenterotoxin of Escherichia coli and having thin layer chromatographicand electrophoretic mobilities different from said biologic heat stableenterotoxin.
 2. The synthetic polypeptide according to claim 1wherein:"e" is zero when "a" is zero, "d" is zero when "b" is zero, and"f" is zero when "c" is zero; each of "g" and "k" is zero when "a" iszero, each of "h" and "j" is zero when "b" is zero, and each of "i" and"l" is zero when "c" is zero; and an intrapolypeptide cystine disulfidebond is present between the Cys residues shown in said formula as bondedto R_(a) ¹ and R_(e) ⁵ or R_(b) ² and R_(d) ⁴ or R_(c) ³ and R_(f) ⁶. 3.The synthetic polypeptide according to claim 2 wherein "b" is zero andan intrapolypeptide cystine disulfide bond is present between the Cysresidues shown in said formula as bonded to R_(b) ² and R_(d) ⁴.
 4. Thesynthetic polypeptide according to claim 2 wherein "b" and "c" are zeroand intrapolypeptide cystine disulfide bonds are present between the Cysresidues shown in said formula as bonded to R_(b) ² and R_(d) ⁴, andR_(e) ³ and R_(f) ⁶.
 5. The synthetic polypeptide according to claim 2wherein "a", "b" and "c" are zero and three intrapolypeptide cystinedisulfide bonds are present between the Cys residues shown in saidformula as bonded to R_(a) ¹ and R_(e) ⁵, R_(b) ² and R_(d) ⁴, and R_(e)³ and R_(f) ⁶.
 6. The synthetic polypeptide according to claim 1 whereinsaid intramolecular cystine disulfide bond is an interpolypeptide bondand said polypeptide is one of a plurality of repeating units of amultimer.
 7. The synthetic polypeptide according to claim 1 containingat least two intrapolypeptide cystine residues formed between the pairsof Cys residues shown in said formula as bonded to groups R_(a) ¹ andR_(e) ⁵, R_(b) ² and R_(d) ⁴, or R_(c) ³ and R_(f) ⁶.
 8. The syntheticpolypeptide according to claim 1 wherein "m" is zero, and R_(m) ¹³ isabsent.
 9. The synthetic multimer according to claim 1 containing abouttwo to about three of said polypeptide repeating units, wherein saidrepeating units are bonded together by said amide bond and saidintramolecular cystine disulfide bond is an intrapolypeptide disulfidebond.
 10. The synthetic multilmer according to claim 1 having an averagemolecular weight of at least about 400,000 daltons, wherein saidrepeating units are bonded together by said intramolecularinterpolypeptide cystine disulfide bonds.
 11. A synthetic polypeptidehaving the amino acid residue sequence, taken from left to right and inthe direction from amino-terminus to carboxy-terminus, represented bythe formula:

    AsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsnAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn

said polypeptide being free of sulfhydryl groups, having at least about10% of the antigenicity of that of biologic heat stable enterotoxin ofEscherichia coli, and thin layer chromatographic and electrophoreticmobilities different from said biologic heat-stable enterotoxin.
 12. Anantigenic synthetic polypeptide including the amino acid residuesequence, taken from left to right and in the direction fromamino-terminus to carboxy-terminus, represented by the formula:

    MetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn

said polypeptide being free of sulfhydryl groups, having at least about10% of the antigenicity of that of biologic heat stable enterotoxin ofEscherichia coli and capable of inducing antibodies to said enterotoxin,and having thin layer chromatographic and electrophoretic mobilitiesdifferent from said biologic heat-stable enteroxtin.
 13. An antigenicsynthetic polypeptide including the amino acid residue sequence, takenfrom left to right and in the direction from amino-terminus tocarboxy-terminus, represented by the formula:

    TyrThrGluSerMetAlaGlyLysArgGluAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn

said polypeptide being free of sulfhydryl groups, having at least about10% of the antigenicity of that of biologic heat stable enterotoxin ofEscherichia coli and capable of inducing antibodies to said enterotoxin,and having thin layer chromatographic and electrophoretic mobilitiesdifferent from said biologic heat-stable enterotoxin.
 14. An antigenicsynthetic polypeptide including the amino acid residue sequence, takenleft to right and in the direction from amino-terminus tocarboxy-terminus, represented by the formula: ##STR19## wherein thethree specific amino acid residues in parentheses are each analternative to the immediately preceding amino acid residue in saidsequence;a-f and g-l are integer each having a value of zero or one,with the proviso that if the value of any of a-f or g-l is zero, thecorresponding R_(a-f) ¹⁻⁶ or R_(g-l) ⁷⁻¹² -group is absent, and when anR_(a-f) ¹⁻⁶ -group is absent the sulfur atom of the respective Cysresidues having an absent R_(a-f) ¹⁻⁶ -group forms a cystine disulfidebond, while if the value of said a-f or g-l is one, said correspondingR_(a-f) ¹⁻⁶ - or R_(g-l) ⁷⁻¹² -group is present; said R_(a-f) ¹⁻⁶-groups when taken individually, are the same or different moietiesbonded to the sulfur atom of the respective Cys residues of the formulaand are selected from the group consisting of hydrogen, an alkyl groupcontaining 1 to about 4 carbon atoms, and a substituted alkyl groupcontaining 2 to about 20 carbon atoms; said R_(g-l) ⁷⁻¹² -groups arealternative Ser amino acid residues to each immediately preceding Cysresidue shown in the formula; at least two of a-f and two of g-l arezero and two Cys residues are present with the proviso that saidsynthetic polypeptide contains at least one intramolecular cystinedisulfide bond formed from at least two Cys residues present; and "m" isan integer having the value of zero or one with the proviso that if "m"is zero R_(m) ¹³ is absent, and if "m" is one, R_(m) ¹³ is a linkinggroup, the acyl portion of a carboxylic acid containing 1 to about 20carbon atoms that forms an amide bond with the amine of theamino-terminal residue, an amino acid residue or a polypeptide chain ofamino acid residues wherein the amino acid residues of said polypeptideare taken from left to right and in the direction from amino-terminus tocarboxy-terminus, and selected from the group consisting of(a) Tyr; (b)##STR20## (c) ##STR21## (d) ##STR22## (d)MetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsn(Ser)Thr(Ser)Phe(Asn)Tyr;(f) TyrThrGluSerMetAlaGlyLysArgGlyAsn(Ser)Thr(Ser)Phe(Asn)Tyr; (g)TyrThrGluSerMetAlaGlyLysArgGlyAsn(Ser)Thr(Ser)Phe(Asn)Tyr; (h)TyrThrGluSerMetAlaGlyLysArgGluMetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsn(Ser)Thr(Ser)Phe(Asn)Tyr;(i)MetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLys;(j) TyrThrGluSerMetAlaGlyLysArgGly; and (k)TyrThrGluSerMetAlaGlyLysArgGluMetValIleIleThrPheMetSerGlyGluThrPheGlnValGluVlProGlySerGlnHisIleAspSerGlnLyswherein the immediately above parenthesized amino acid residues, R_(a-f)¹⁻⁶ and R_(g-l) ⁷⁻¹² are as before defined; said polypeptide having atleast about 10% of the antigenicity of that of biologic heat-stableenterotoxin of Escherichia coli and capable of inducing antibodies tosaid enterotoxin, and having thin layer chromatographic andelectrophoretic mobilities different from said biologic heat stableenterotoxin.
 15. An antigenic synthetic polypeptide including the aminoacid sequence taken from left to right in the direction fromamino-terminus to carboxy-terminus, represented by the formula:##STR23## wherein lines connecting two Cys residues representintramolecular disulfide bonds of cystine residues;the six specificamino acid residues in parentheses are each an alternative to theimmediately preceding amino acid residue; R_(m) ¹³ is a linking group,the acyl portion of a carboxylic acid containing 1 to about 20 carbonatoms that forms an amide bond with the amine of the amino-terminalresidue, or a polypeptide corresponding in sequence to a polypeptide,taken from left to right and in the direction from amino-terminus tocarboxy-terminus, selected from the group consistingofMetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLys;TyrThrGluSerMettAlaGlyLysArgGly; andTyrThrGlusSerMetAlaGlyLysARgGluMetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLyssaid polypeptide having at least about 10% of the antigenicity of thatof biologic heat-stable enterotoxin of Escherichia coli and capable ofinducing antibodies to said enterotoxin, and having thin layerchromatographic and electrophoretic mobilities different from saidbiologic heat stable enterotoxin.
 16. An antigenic synthetic networkpolymer having at least about 10% of the antigenicity of that ofheat-stable enterotoxin of Escherichia coli and capable of inducingantibodies to said enterotoxin comprising a plurality of polypeptiderepeating units including the amino acid residue sequence, taken left toright and in the direction from amino-terminus to carboxy-terminus,represented by the formula: ##STR24## wherein the three specific aminoacid residues in parentheses are each an alternative to the immediatelypreceding amino acid residue in said sequence;a-f and g-l are integerseach having a value of zero or one, with the proviso that if the valueof any of a-f or g-l is zero, the corresponding R_(a-f) ¹⁻⁶ - or R_(g-l)⁷⁻¹² -group is absent, and when an R_(a-f) ¹⁻⁶ -group is absent thesulfur atom of the Cys residue having an absent R_(1-f) ¹⁻⁶ -group formsa cystine disulfide bond, while if the value of said a-f or g-l is one,said corresponding R_(a-f) ¹⁻⁶ - or R_(g-l) ⁷⁻¹² - group is present;said R_(a-f) ¹⁻⁶ groups when taken individually, are the same ordifferent moieties bonded to the sulfur atom of the Cys residue and areselected from the group consisting of hydrogen, an alkyl groupcontaining 1 to about 4 carbon atoms, and a substituted alkyl groupcontaining 2 to about 20 carbon atoms; said R_(g-l) ⁷⁻¹² groups arealternative Ser amino acid residues to each immediately preceding Cysresidue; at least two of a-f and two of g-l are zero and two Cysresidues are present with the proviso that said synthetic polypeptidecontains at last one intramolecular cystine disulfide bond formed fromthe at least two Cys residues present; and "m" is an integer having thevalue of zero or one with the proviso that if "m" is zero R_(m) ¹³ isabsent, and if "m" is one, R_(m) ¹³ is a linking group, the acyl portionof a carboxylic acid containing 1 to about 20 carbon atoms that forms anamide bond with the amine of the amino-terminal residue, an amino acidresidue or a polypeptide chain of amino acid residues wherein the aminoacid residues of said polypeptide are taken from left to right and inthe direction from amino-terminus to carboxy-terminus, and selected fromthe group consisting of(a) Tyr; (b) Asn(Ser)Thr(Ser)Phe(Asn)Tyr; (c)##STR25## (d) ##STR26## (e) ##STR27## (f)MetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsn(Ser)Thr(Ser)Phe(Asn)Tyr;(g) TyrThrGluSerMetAlaGlyLysArgGlyAsn(Ser)Thr(Ser)Phe(Asn)Tyr; (h)TyrThrGluSerMetAlaGlyLysArgGluMetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsn(Ser)Thr(Ser)Phe(Asn)Tyr;(i)MetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLys;(j) TyrThrGluSerMetAlaGlyLysArgGly; and (k)TyrThrGluSerMetAlaGlyLysArgGluMetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLyswherein the immediately above parenthesized amino acid residues, R_(a-f)¹⁻⁶ and R_(g-l) ⁷⁻¹² are as before defined; said plurality of repeatingunits being bonded together by cross-links supplied by intramolecular,interpolypeptide cystine disulfide bonds.
 17. An antigenic syntheticnetwork polymer comprising a plurality of polypeptide repeating units,said synthetic polymer having at least about 10% of the antigenicity ofthat of biologic heat-stable enterotoxin of Escherichia coli and capableof inducing antibodies to said enterotoxin, having thin layerchromatographic and electrophoretic mobilities different from saidbiologic heat-stable enterotoxin, being free of sulfhydryl group andhaving an average molecular weight of at least about 40,000 daltons,said repeating units including the amino acid residue sequence, takenfrom left to right and in the direction from amino-terminus tocarboxy-terminus, represented by the formula:

    Asn(Ser)Thr(Ser)Phe(Asn)TyrCysCysGluLeuCysCys

    Tyr(Asn)ProAla(Thr)CysAlaGlyCysAsn(Tyr)

wherein the six specific amino acid residues in parentheses are each analternative to the immediately preceding amino acid residue; saidrepeating units being bonded together by intramolecular,interpolypeptide cystine disulfide bonds formed between the Cys residuesof said polypeptide repeating units.
 18. The antigenic network polymeraccording to claim 17 wherein said polypeptide repeating unit includesthe amino acid residue sequence, taken from left to right and in thedirection from amino-terminus to carboxy-terminus, represented by theformula:

    AsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn.


19. The synthetic network polymer according to claim 16 wherein:all ofsaid a-f are zero; all of said g-l are zero; said "m" is one; and saidR_(m) ¹³ is a peptide containing the amino acid residue sequence of thefour amino-terminal residues of ST Ib.